Apparatus, system and method for performing automated centrifugal separation

ABSTRACT

Systems, methods and devices are provided for the automated centrifugal processing of samples. In some embodiments, an integrated fluidic processing cartridge is provided, in which a centrifugation chamber is fluidically interfaced, through a lateral surface thereof, with a microfluidic device, and wherein the integrated fluidic processing cartridge is configured to be inserted into a centrifuge for centrifugation. A cartridge interfacing assembly may be employed to interface with the integrated fluidic processing cartridge for performing various fluidic processing steps, such as controlling the flow of fluids into and out of the centrifugation chamber, and controlling the flow of fluids into the microfluidic device, and optionally for the further fluidic processing of fluids extracted to the micro-fluidic device. The integrated fluidic processing cartridge may include a supernatant chamber the extraction of a supernatant thereto, and a diluent chamber for diluting a suspension collected in the centrifugation chamber.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No.61/994,728, titled “APPARATUS, SYSTEM AND METHOD FOR PERFORMINGAUTOMATED CENTRIFUGAL SEPARATION” and filed on May 16, 2014, the entirecontents of which is incorporated herein by reference.

BACKGROUND

The present disclosure relates to sample preparation, centrifugalseparation, and microfluidic processing of fluids.

Pathogen detection in whole blood samples using molecular techniquesrequires a sample treatment process which yields a suspension of targetnucleic acids which is sufficiently free of PCR inhibitors, interferentsand non-target nucleic acids. The sample treatment process is closelytied to the amplification and detection techniques utilized and as suchare vital to sensitive and specific detection of target microbes. Forinstance, the number of target microbial cells in whole blood, on theorder of 10¹ CFU/mL, is vastly outnumbered by blood cells, on the orderof 10¹⁰/mL. Blood cells are therefore sources of large amount ofbackground DNA, PCR inhibitors, RNase, and fluorescence quenchers.Moreover, dead microbes and nucleic acid from such microbes may also bepresent in the sample from previously treated infections. This imposesstrict functionality requirements on the nucleic acid based pathogendetection platforms.

Existing methods of performing sample preparation on whole blood samplestypically consist of the following steps: (i) the blood sample issubjected to some means of lysing the blood cells and microbial cells,either selectively or non-selectively with respect to the targetmicrobes; (ii) removal or inactivation of inhibitors and interferents toPCR and detection; and (iii) removal of non-target nucleic acid orenhanced amplification and detection strategies for increasingspecificity with respect to target microbes and live versus deadmicrobes.

These steps are typically performed either separately or in combinationand with varying levels of efficacy in accordance with the tolerancecharacteristics of downstream processes. Most existing pathogendetection platforms rely on extraction and purification of the targetnucleic acids prior to amplification and detection using PCR or RT-PCR,and are poorly-suited for automation in applications involving lowpathogen concentrations.

SUMMARY

Systems, methods and devices are provided for the automated centrifugalprocessing of samples. In some embodiments, an integrated fluidicprocessing cartridge is provided, in which a centrifugation chamber isfluidically interfaced, through a lateral surface thereof, with amicrofluidic device, and wherein the integrated fluidic processingcartridge is configured to be inserted into a centrifuge forcentrifugation. A cartridge interfacing assembly may be employed tointerface with the integrated fluidic processing cartridge forperforming various fluidic processing steps, such as controlling theflow of fluids into and out of the centrifugation chamber, andcontrolling the flow of fluids into the microfluidic device, andoptionally for the further fluidic processing of fluids extracted to themicrofluidic device. The integrated fluidic processing cartridge mayinclude a supernatant chamber the extraction of a supernatant thereto,and a diluent chamber for diluting a suspension collected in thecentrifugation chamber.

Accordingly, in one aspect, there is provided a method of performingcentrifugal separation and microfluidic processing using an integratedfluidic processing cartridge;

the integrated fluidic processing cartridge comprising:

-   -   a macrofluidic centrifugation chamber, wherein a distal region        of the macrofluidic centrifugation chamber is configured to        collect a sediment under the application of centrifugal force;    -   a microfluidic device having an inner surface and an outer        surface, wherein the inner surface is attached to a lateral        surface of the macrofluidic centrifugation chamber, and wherein        the microfluidic device comprises one or more fluidic components        that are configured to be actuated through the outer surface;    -   wherein a sediment extraction port is provided within the        macrofluidic centrifugation chamber and wherein the sediment        extraction port is in fluid communication, through the lateral        surface, with a sediment extraction channel of the microfluidic        device for extraction of the sediment to the microfluidic        device;

the method comprising:

-   -   providing a liquid sample within the macrofluidic centrifugation        chamber;    -   centrifuging the integrated fluidic processing cartridge with a        centrifugation device such that the sediment is collected within        the distal region;    -   applying a pressure difference between the sediment extraction        channel of the microfluidic device and the macrofluidic        centrifugation chamber, such that a concentrated suspension        comprising at least a portion of the sediment flows through the        sediment extraction port and into the microfluidic device,        thereby transferring the concentrated suspension to the        microfluidic device; and    -   fluidically processing the concentrated suspension within the        microfluidic device by actuating one or more of the fluidic        components through the outer surface.

In another aspect, there is provided a system for performing centrifugalseparation and microfluidic processing, said system comprising:

an integrated fluidic processing cartridge comprising:

-   -   a macrofluidic centrifugation chamber, wherein a distal region        of said macrofluidic centrifugation chamber is configured to        collect a sediment under the application of centrifugal force;    -   a microfluidic device having an inner surface and an outer        surface, wherein said inner surface is attached to a lateral        surface of said macrofluidic centrifugation chamber, and wherein        said microfluidic device comprises one or more fluidic        components that are configured to be actuated through said outer        surface;    -   wherein a sediment extraction port is provided within said        macrofluidic centrifugation chamber and wherein said sediment        extraction port is in fluid communication, through said lateral        surface, with a sediment extraction channel of said microfluidic        device for extracting the sediment thereto; and

a centrifugation device comprising:

-   -   a rotor; and    -   a receptacle pivotally connected to said rotor, wherein said        receptacle is configured to receive said integrated fluidic        processing cartridge such that said outer surface is laterally        and outwardly oriented relative to a rotational axis of said        rotor when said rotor is at rest;

a cartridge interfacing assembly configured to be removably interfacedwith said integrated fluidic processing cartridge when said rotor is atrest: and

a control and processing unit operably interfaced with thecentrifugation device and the cartridge interfacing assembly, whereinsaid control and processing unit is configured to:

-   -   control said centrifugation device to centrifuge said integrated        fluidic processing cartridge;    -   control said cartridge interfacing assembly to interface said        cartridge interfacing assembly with said integrated fluidic        processing cartridge when said centrifugation device is at rest;    -   control said cartridge interfacing assembly to actuate the        application of a pressure difference between said macrofluidic        centrifugation chamber and said sediment extraction channel to        extract, onto the microfluidic device, a concentrated suspension        comprising at least a portion of the sediment; and to        fluidically process the concentrated suspension on the        microfluidic device.    -   control said cartridge interfacing assembly to actuate the one        or more fluidic components to fluidically process the        concentrated suspension on the microfluidic device.

In another aspect, there is provided an integrated fluidic processingcartridge for performing macrofluidic separation and microfluidicprocessing, said integrated fluidic processing cartridge comprising:

a macrofluidic centrifugation chamber, wherein a distal region of saidmacrofluidic centrifugation chamber is configured to collect a sedimentunder the application of centrifugal force;

a microfluidic device having an inner surface and an outer surface,wherein said inner surface is attached to a lateral surface of saidmacrofluidic centrifugation chamber, and wherein said microfluidicdevice comprises one or more fluidic components that are configured tobe actuated through said outer surface;

wherein a sediment extraction port is provided within said distal regionof said macrofluidic centrifugation chamber, and wherein said sedimentextraction port is in fluid communication, through said lateral surface,with a sediment extraction channel of said microfluidic device, forextracting the sediment thereto.

In another aspect, there is provided a microfluidic diaphragm valvecomprising:

a base layer having a port formed in a surface thereof;

a microfluidic layer having a first surface and an opposing secondsurface, wherein said microfluidic layer is provided on said base layersuch that said second surface is attached to said surface of said baselayer,

said microfluidic layer comprising a lateral microfluidic channel influid communication with a valve seat aperture, wherein said valve seataperture is positioned over said port, and wherein said valve seataperture extends through said microfluidic layer;

a membrane adhered to said second surface of said microfluidic layer,said membrane enclosing said valve seat aperture; and

a plunger positioned to contact an external surface of said membrane,such that upon application of a sufficient inwardly directed force tosaid plunger, said plunger is received within said valve seat apertureand said membrane forms a seal against said port.

In another aspect, there is provided a method of performing centrifugalseparation using an integrated fluidic processing cartridge;

the integrated fluidic processing cartridge comprising:

-   -   a macrofluidic centrifugation chamber, wherein a distal region        of the macrofluidic centrifugation chamber is configured to        collect a sediment under the application of centrifugal force,        and wherein a supernatant extraction port is provided within the        distal region of the macrofluidic centrifugation chamber;    -   a supernatant chamber having a supernatant delivery port formed        therein; and    -   a microfluidic device having an inner surface and an outer        surface, wherein the inner surface is attached to a lateral        surface of the macrofluidic centrifugation chamber;    -   wherein the supernatant extraction port is in fluid        communication, through the lateral surface, with a supernatant        delivery channel of the microfluidic device, and wherein the        supernatant delivery port is in fluid communication, through the        lateral surface, with the supernatant delivery channel of the        microfluidic device for extracting a substantial portion of a        supernatant from the macrofluidic centrifugation chamber into        the supernatant chamber; and

the method comprising:

-   -   providing a liquid sample within the macrofluidic centrifugation        chamber;    -   centrifuging the integrated fluidic processing cartridge with a        centrifugation device such that the sediment is collected within        the distal region;    -   applying a pressure difference between the supernatant chamber        and the macrofluidic centrifugation chamber, such that the        supernatant flows through the supernatant delivery channel,        thereby transferring the supernatant to the supernatant chamber.

A further understanding of the functional and advantageous aspects ofthe disclosure can be realized by reference to the following detaileddescription and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments will now be described, by way of example only, withreference to the drawings, in which:

FIG. 1 shows a schematic of an example system for performing automatedcentrifugation and washing with an integrated fluidic processingcartridge.

FIGS. 2A-C show different views of an example integrated fluidicprocessing cartridge for centrifugation and washing.

FIG. 2D illustrates an example implementation of an integrated fluidiccartridge that is suitable for performing supernatant extraction duringcentrifugation.

FIG. 3 provides a flow chart illustrating an example method forperforming automated centrifugation and washing.

FIGS. 4A and 4B provide front views of an embodiment of an exampleintegrated fluidic processing cartridge including a port for extractionof the sedimented particles, or a suspension thereof.

FIG. 5 is an illustration of an example integrated fluidic processingcartridge configured for extraction of a sample directly from acollection tube, and subsequent centrifugation and washing, to obtain aconcentrated and purified suspension of microbial cells.

FIG. 6 shows schematic cross-sectional views of a channel which has beenequipped with a filter intended for retaining cells.

FIG. 7A is a flow chart describing a method of sample preparation,electrical lysis, and multiplexed molecular detection of nucleic acidspresent in the lysate, according to one example embodiment of thepresent disclosure.

FIG. 7B is a flow chart describing a method of sample preparationaccording to one example embodiment of the present disclosure.

FIG. 7C is a flow chart describing a method of sample preparation andelectrical lysis according to one example embodiment of the presentdisclosure.

FIG. 7D is a flow chart describing a method of sample preparation,electrical lysis, and protein extraction, optionally for subsequentlyMALDI-TOF analysis, according to one example embodiment of the presentdisclosure.

FIG. 8 is a schematic of a portion of an example integrated fluidicprocessing cartridge, in which additional fluidic components areprovided for processing of separated and concentrated microbial cells.

FIGS. 9A-B illustrate various example embodiments of a thermal chamber.

FIG. 9C is an illustration of an example heater element for use with athermal chamber.

FIGS. 9D-E illustrate various example embodiments of an array of thermalchambers.

FIG. 10A is an illustration of an example integrated fluidic processingcartridge, showing front and back lateral surfaces from an isometricview.

FIG. 10B is an illustration of an example integrated fluidic processingcartridge, showing an exploded view.

FIG. 10C-K is an illustration of an example multi-laminate integratedfluidic processing cartridge, showing detail of the major layers.

FIGS. 11A-I provide illustrations of example embodiments of a valve andassociated plunger.

FIGS. 12A-B illustrate of an example embodiment of a port and associatedair displacement mechanism, showing (A) cross-sectional and (B) overheadviews.

FIG. 13A illustrates the insertion of an integrated fluidic processingcartridge into a receptacle.

FIGS. 13B and 13C illustrate a hanging bucket centrifuge according to anexample embodiment.

FIGS. 14A and 14B illustrate an example embodiment involving theengagement of a cartridge interfacing assembly with an integratedfluidic processing cartridge housed in a rotor.

FIGS. 15A-E illustrate alternative example embodiments for actuating avalve plunger.

FIG. 16 illustrates an example implementation of a mechanism forvortexing the integrated fluidic processing cartridge via orbitalmotion.

FIG. 17A-C illustrate example valve latching mechanisms.

FIG. 18A illustrates an example implementation of an integrated fluidicprocessing cartridge supported in a receptacle.

FIGS. 18B-C illustrate an example implementation of a cartridgeinterfacing assembly, where FIG. 18C illustrates the engagement of thecartridge interfacing assembly with the receptacle supporting theintegrated fluidic processing device.

FIG. 19 illustrates an example optical system that can be optionallyintegrated with the cartridge interfacing assembly.

DETAILED DESCRIPTION

Various embodiments and aspects of the disclosure will be described withreference to details discussed below. The following description anddrawings are illustrative of the disclosure and are not to be construedas limiting the disclosure. Numerous specific details are described toprovide a thorough understanding of various embodiments of the presentdisclosure. However, in certain instances, well-known or conventionaldetails are not described in order to provide a concise discussion ofembodiments of the present disclosure.

As used herein, the terms “comprises” and “comprising” are to beconstrued as being inclusive and open ended, and not exclusive.Specifically, when used in the specification and claims, the terms“comprises” and “comprising” and variations thereof mean the specifiedfeatures, steps or components are included. These terms are not to beinterpreted to exclude the presence of other features, steps orcomponents.

As used herein, the term “exemplary” means “serving as an example,instance, or illustration,” and should not be construed as preferred oradvantageous over other configurations disclosed herein.

As used herein, the terms “about” and “approximately” are meant to covervariations that may exist in the upper and lower limits of the ranges ofvalues, such as variations in properties, parameters, and dimensions.Unless otherwise specified, the terms “about” and “approximately” meanplus or minus 25 percent or less.

It is to be understood that unless otherwise specified, any specifiedrange or group is as a shorthand way of referring to each and everymember of a range or group individually, as well as each and everypossible sub-range or sub -group encompassed therein and similarly withrespect to any sub-ranges or sub-groups therein. Unless otherwisespecified, the present disclosure relates to and explicitly incorporateseach and every specific member and combination of sub-ranges orsub-groups.

As used herein, the term “on the order of”, when used in conjunctionwith a quantity or parameter, refers to a range spanning approximatelyone tenth to ten times the stated quantity or parameter.

Unless defined otherwise, all technical and scientific terms used hereinare intended to have the same meaning as commonly understood to one ofordinary skill in the art. Unless otherwise indicated, such as throughcontext, as used herein, the following terms are intended to have thefollowing meanings:

As used herein, the phrase “centrifugal separation” refers to a processof centrifugation of a sample fluid containing particulate or solidmaterial, whereby sedimentation of such particulate or solid materialsoccurs, thereby producing a sediment. The phrase “sediment” generallyrefers to one or more particles that are collected, within a distalregion of a centrifugation device, after the application of acentrifugal force. One non-limiting example of a sediment is one or moremicrobial cells. A sediment need not be collected at the bottom surfaceof a centrifugation chamber, and may instead be formed near the bottomof the centrifugation chamber, or at the interface between a supernatantand a cushioning liquid, as described in detail herebelow.

As used herein, the phrases “wash” and “washing” refers to a processinvolving the addition of a diluent (or wash liquid/buffer) to a solidor suspension sample, mixing of the diluent with the sample (optionallyto re-suspend a sediment) to obtain a suspension, and centrifuging thesuspension.

As used herein, the term “microfluidic channel” refers to a fluidicchannel having a cross-sectional dimension less than 1 mm.

As used herein, the term “microfluidic device” refers to a fluidicdevice having at least one microfluidic channel.

As used herein, the term “macrofluidic chamber” refers to a fluidicchamber or chamber, where all dimensions of the fluidic chamber orchamber exceed 1 mm, and where a volume of the chamber exceeds 500microliters.

Integrated Apparatus for Centrifugation and Washing

Referring now to FIG. 1, an illustration is provided of an exampleintegrated system 100 for performing automated centrifugal separation orautomated centrifugal separation with washing. Example system 100includes centrifuge 110, which receives one or more integrated fluidicprocessing cartridges 120 for centrifugal separation. Centrifuge 110includes one or more receptacles 112 which are connected to a motorizedrotor 114 and are configured to receive integrated fluidic processingcartridges 120. The cartridge receptacles 112 may be, for example, ofthe fixed angle type or the swinging bucket type which are common inlaboratory centrifuges (e.g. each receptacle 112 may be pivotallyconnected to the motorized rotor 114).

Cartridge interface assembly (unit) 130 is configured to removablyengage (or interface) with an integrated fluidic processing cartridge120 when the motorized rotor 114 is at rest, for controlling the flow offluids within integrated fluidic processing cartridge 120. Theinterfacing of the cartridge interfacing assembly with the integratedfluidic cartridge may occur, for example, via a direct interface betweenthe cartridge interfacing assembly and the integrated fluidic cartridge120, or, for example, via an interface (e.g. an actuation interface) onthe centrifuge 110 (e.g. on the motorized rotor 114 or cartridgereceptacle 112). Centrifuge 110 and cartridge interfacing assembly 130are controlled via control and processing unit 140.

As described in further detail below, each integrated fluidic processingcartridge 120 includes a centrifugation chamber for centrifugalseparation during rotation of the motorized rotor 114. In someembodiments, the centrifugation chamber may be a microfluidic chamber.In various example embodiments described below, the centrifugationchamber is a macrofluidic centrifugation chamber capable of performingcentrifugal separations for fluid volumes exceeding 500 microliters.

Integrated fluidic processing cartridge 120 may contain ports, conduits,valves and chambers to enable removal of the supernatant from themacrofluidic centrifugation chamber and optionally storage of theremoved supernatant on the cartridge while integrated fluidic processingcartridge 120 is housed within the centrifuge 110. Integrated fluidicprocessing cartridge 120 may also include ports, conduits, valves, andchambers to enable automated washing while integrated fluidic processingcartridge 120 is housed within centrifuge 110.

An illustration of an example embodiment of integrated fluidicprocessing cartridge 120 is shown in FIGS. 2A-C, where FIGS. 2A, 2B and2C show top, front and back views respectively (FIG. 2C shows the outerlateral surface of the device). In this embodiment integrated fluidicprocessing cartridge 120 includes macrofluidic centrifugation chamber200, diluent chamber 210 and supernatant chamber 220. In the exampleembodiment shown, macrofluidic centrifugation chamber 200 has a conicalor round bottom shape, and a smooth inner surface in order to minimizeadsorption or trapping of the sedimented particulate matter duringcentrifugation. The centrifugation chamber 200 is oriented in thecentrifuge rotor 110 such that the centrifugal force acts in thedirection of the conical or round bottom of the chamber. Diluent chamber210 includes a diluent liquid, whose composition is selected to conformto the requirements of the final medium into which the particles will beresuspended which may be dictated by subsequent processing requirements.The diluent chamber 210 may have a conical or narrowing bottom tip thatenables the extraction of wash liquid with minimal residual. One or moreadditional diluent chambers may be included together with the requiredconduits, and valves, to enable one or more diluent liquids of differentcompositions to be used in the wash process. Supernatant chamber 220 maybe empty, or may include an adsorbent material, such as a wickingmaterial. Supernatant chamber may be employed to collect thesupernatant, and/or employed as a waste chamber.

Automated separation of a suspension is performed within macrofluidiccentrifugation chamber 200 by performing centrifugal sedimentation ofthe particulate material in the suspension followed by flowing ofsupernatant from macrofluidic centrifugation chamber 200 to supernatantchamber 220. One or more washes may be performed by the additionalsequential steps of flowing a diluent liquid from diluent chamber 210into macrofluidic centrifugation chamber 200, optionally mixing thediluent and the residual supernatant, performing centrifugation, andflowing supernatant from macrofluidic centrifugation chamber 200 tosupernatant chamber 220. As described in additional embodiments that areprovided below, integrated fluidic processing cartridge may includeadditional features and components, such as, but not limited to, alysing chamber and/or one or more assay chambers or wells for subsequentprocessing and/or assaying of the washed sample.

In some example embodiments of the present disclosure, the integratedfluidic processing cartridge is configured to support automatedcentrifugal separation and optionally dilution/washing in a closedconfiguration. In the present disclosure, the term “closedconfiguration” refers to a cartridge structure that prevents theaddition or removal of liquids from the cartridge during fluidicprocessing. Whereas various example embodiments may employ vents and theinjection of air (or other gases) into the cartridge or the evacuationof air (or other gases) from the cartridge, gas permeable membranes orfilters of sufficiently small pore size may be placed in such air pathsto prevent or minimize the egress of hazardous particles or fluids andthe ingress of contaminants or interferents.

In the example embodiment illustrated in FIGS. 2A-2C, the chambers areinterfaced with a microfluidic device 205, which has internal fluidicchannels that provide fluidic connections between the chambers. Invarious example embodiments described below, the microfluidic device 205includes one or more microfluidic layers, and has an inner surface andan outer surface. In the present example embodiment, the inner surfaceis attached to lateral surfaces of the macrofluidic centrifugationchamber, the diluent chamber 210, and the supernatant chamber 220, asshown in FIGS. 2A and 2B. The microfluidic device 205 is in fluidcommunication with the chambers through ports (holes, apertures or vias)formed through the walls of the chambers. Microfluidic device 205includes channels (which may be microfluidic channels) for fluid flowbetween chambers and ports, for allowing fluidic movements into or outof chambers. Microfluidic device 205 may also include one or more valvesfor controlling fluid flow. Fluid flow may be produced by any suitableflow mechanism. In one example implementation, fluid flow betweendifferent locations within the integrated fluidic processing cartridgeis produced using interfaces to a gas (e.g. air) displacement devicethat generates a pressure difference between chambers, forgas-displacement induced fluidic movements.

In one example implementation, the chambers 200, 210 and 220 may beformed from plastic material (such as, but not limited to,polycarbonate, polypropylene, PET, polystyrene, cyclic olefins,acrylics, polyethylene, polyurethanes, PTFE, PEEK, PVC), eitherindividually or in combination, using a fabrication process such asinjection molding, casting, machining, 3D printing or other methods andmaterials known to those skilled in the art. In addition to the formingprocess, some or all of the chambers 200, 210 or 220 may optionally needfurther finishing or surface treatment to ensure a smooth low bindingsurface. Such finishing may be carried out by using various processeseither individually, or in combination such as mechanical polishing, orchemical coating of the inner surface by silicones, nonionic silanes,treatment to cause the surface to by hydrophobic, treatment to cause thesurface to be hydrophilic, BSA, PEG, SAMs or other similar compounds,via dip coating processes, spray coating, or other methods known tothose skilled in the art with or without following curing steps asrequired by the process used. The chambers may be formed to possess aback surface, each chamber having one or more ports formed therein,which interface with fluidic channels of the microfluidic device 205through the inner surface of the microfluidic device.

In one example embodiment that is intended for the detection of pathogenmicrobial cells in whole blood (or, for example, blood added to culturemedium), the volume of centrifugation, dilution and supernatant chambersmay be, respectively, in the range of 0.1-60 mL, 0.5-120 mL, and 0.6-120mL. The more preferred examples ranges for the diluent and supernatantchambers are, respectively, 0.5-10 mL, 1.5-20 mL, and 1.5-20 mL.According to various example implementations, depending on the geometryand size of the chambers, the diameters of their associated holes, portsand vents may be selected to be in the 0.1 mm-3 mm range. According tovarious example implementations, the width of the conduits may vary in0.1 mm to 3 mm range. According to various example implementations, theheight of the conduits may vary in 0.025 mm to 1 mm range.

In one example implementation, the microfluidic device 205 may be alaminate structure formed from multiple layers which contain the fluidicchannels (conduits), chambers, and fluidic components that may beexternally actuated, such as valves and gas permeable interfaces thatmay be employed for fluid control.

The microfluidic device may be formed via a wide variety of fabricationprocesses. Non-limiting examples of fabrication processes includeinjection molding, hot-embossing, micromachining, punching, die cutting,soft lithography, laser cutting, water jet cutting, plotting cutters orother methods know to those skilled in the art. Layers may be made withmaterials such as, but not limited to, polycarbonate, PET,polypropylene, PDMS, cyclic olefins, PMMA, photoresists, silicon wafers,glass, foils such as aluminum or other materials which are known tothose skilled in the art.

The microfluidic device 205 may be formed by lamination of itsconstituent layers by methods such as, but not limited to, adhesivebonding, thermal bonding, ultrasonic bonding, or other bonding methodsknown to those in the art. In addition some or all of the layers mayoptionally require surface treatment to provide additional propertiessuch as low energy non-binding, enhanced hydrophilic or enhancedhydrophobic properties to prevent adhesion of compounds within thesample to the walls of the device, or allow ease of fluid passage in thechambers or channels, or act as passive fluid control elements, as isknown to those skilled in the art. These properties can be establishedby chemical treatment of the materials with compounds such as, but notlimited to, silicones, silanes, PEG, BSA or other materials known tothose skilled in the art. The inner surface of the microfluidic device205 may be bonded to the lateral surface of the chambers to form theintegrated fluidic processing cartridge 120. In some embodiments, theback surfaces of the chambers are co-planar, and the inner surface ofthe microfluidic device 205 is a planar surface.

In an alternative example embodiment, some or all of the fluidiccomponents of the microfluidic device 205 may be integrally formed withthe chambers, thereby forming an intermediate device having a lateralsurface, and any remaining layers of the microfluidic device 205 may bebonded to the lateral surface to form the integrated fluidic processingcartridge 120.

In other example embodiments, one or more of the supernatant chamber andthe diluent chamber (or a plurality thereof) may be externally providedrelative to the integrated fluidic processing cartridge, and externallyinterfaced thereto. For example, the ports provided on the lateralsurface of the centrifugation chamber may include fluidic connectorsthat are suitable for forming a fluidic connection (directly orindirectly) with one or more external diluent chambers, supernatantchambers, or other external fluidic reservoirs (for example, an externallysis buffer, external reagent, and/or external growth medium). In oneexample implementation, one or more external chambers may be provided on(e.g. housed within or received within) the cartridge interfacingassembly, such that the one or more external chambers may be removablyfluidically interfaced with the integrated fluidic processing cartridge.In one example implementation, a port within the macrofluidiccentrifugation chamber may be fluidically interfaced with themicrofluidic device, as described above, and the microfluidic device mayinclude a fluidic connector, such that the macrofluidic centrifugationchamber is brought into fluid communication with the external chamberthrough the microfluidic device. In such an example embodiment, themicrofluidic device may include one or more valves for optionallyrestricting the flow of fluid between the macrofluidic centrifugationchamber and the external chamber.

Referring again to the non-limiting example embodiment illustrated inFIGS. 2A-C, diluent chamber 210 and supernatant chamber 220 are eachconnected, through microfluidic device 205, to macrofluidiccentrifugation chamber 200 via diluent delivery channel 230 andsupernatant delivery channel 240, respectively. Diluent delivery channel230 is fluidically connected to diluent delivery port 252 formed in thelateral wall of the macrofluidic centrifugation chamber 200 and todiluent extraction port 251 formed in diluent chamber 210 for thedelivery of diluent from the diluent chamber 210 to the macrofluidiccentrifugation chamber 200. Similarly, supernatant delivery channel 240is fluidically connected supernatant extraction port 256 and supernatantdelivery port 257 for the extraction of supernatant from themacrofluidic centrifugation chamber 210 to the supernatant chamber 220.

In the example embodiment illustrated in FIGS. 2A-C, diluent chamber 210and supernatant chamber 220 each also contain (optionally pierceable)vents 270 and 275, respectively, which are housed in microfluidic device205 and vent to atmospheric pressure though a surface of the integratedfluidic processing cartridge. In one example implementation, one thevents may be accessible through an outer lateral surface of themicrofluidic device 205. In another example implementation, one or moreof the vents may be accessible through a lateral surface of therespective chamber, where the vent is located in a portion of thelateral surface that is not attached to the microfluidic device. Inanother example implementation, one or more of the vents may beaccessible through an upper or lower surface of the respective chamberin which the vent is located (as opposed to through a lateral surface).The chambers 210 and 220 also contain ports 251 and 257 respectively andmay otherwise be closed. Macrofluidic centrifugation chamber 200 is alsoin fluidic communication with port 260 that is housed in microfluidicdevice 205 and accessible through a surface of the integrated fluidicprocessing device. Macrofluidic centrifugation chamber 200 may alsocontain ports 256 and 252 and may be otherwise closed.

In one example implementation, a pressure difference may be appliedtogether with coordinated actuation of valves in order to effect liquidtransfer to and from chambers of the integrated fluidic processingdevice. Flow in diluent delivery channel 230 may be controlled bydiluent control valve 250 which may be located at any position alongdiluent delivery channel 230, but may be preferentially located proximalto diluent extraction port 251. Flow in supernatant delivery channel 240may be controlled by supernatant control valve 255 which may be locatedat any position along supernatant delivery channel 240, but may bepreferentially located proximal to supernatant extraction port 256.Valves 250 and 255 may be actuated through the outer surface ofmicrofluidic device 205, as shown in FIG. 2C.

In one example implementation, the transfer of diluent from diluentchamber 210 to macrofluidic centrifugation chamber 200 may be achievedby selectively opening diluent control valve 250 and selectivelyapplying a negative differential pressure at port 260 relative todiluent chamber 210. Similarly, the transfer of liquid from macrofluidiccentrifugation chamber 200 to supernatant chamber 220 may be achieved byselectively opening supernatant control valve 255 and selectivelyapplying a positive differential pressure at port 260 relative tosupernatant chamber 220. Accordingly, the movement of liquid within theintegrated fluidic cartridge may be controlled by the application of apositive or negative gauge pressure at the port 260 within themacrofluidic centrifugation chamber in combination with the selectiveactuation of the valves between the macrofluidic centrifugation chamberand the various chambers.

In an alternative embodiment, vents 270 and 275 may be configured asports at which an air displacement device (e.g. or a gas displacementdevice) can be engaged and port 260 may be configured as an air vent.The transfer of liquid from chamber 210 to chamber 200 may in this caseperformed by applying a positive differential pressure at port 270relative to chamber 200, and liquid transfer from chamber 200 to chamber220 is performed by applying a negative differential pressure at port275 relative to chamber 200. Valves 250 and 255 are open during theserespective liquid transfer operations and may optionally be omitted fromthe integrated fluidic processing cartridge if not required for otherreasons or modes of operation described herein.

The air displacement device (gas displacement device), which isconnected fluidically to port 260 (and/or optionally configured tointerface with ports 270 or 275) may be, for example, a syringe pump,peristaltic pump, bellows pump or any other pump or air displacementdevice which can controllably deliver or remove air from the cartridgevia the connected port. It will be understood that the air displacementdevices described in the example embodiments provided below may employair, or any other gas, in order to induce the flow of liquids due to theestablishment of a pressure differential between different portions ofintegrated fluidic processing cartridge 120. For example, in someembodiments, a gas source may be interfaced with a pressurization device(e.g. a pump) in order to control the flow of liquids.

In some embodiments, the opening of the valves, and the application of apressure differential between port 260 and vents 270 or 275, isperformed when integrated fluidic processing cartridge 120 is at restand under control of cartridge interfacing assembly 130, which mayselectively engage with the outer surface of microfluidic device 205when integrated fluidic processing cartridge 120 is housed within thecentrifugation device, as described in further detail below. In suchcases, valves 250 and 255 may be configured to be in a closedconfiguration when cartridge interfacing assembly 130 is disengaged andwhen centrifuge 110 is performing centrifugation (examples of suchvalves are provided below).

An additional valve (not shown) may be provided on the fluid pathbetween port 260 and macrofluidic centrifugation chamber 200 in order toprevent fluid from entering the air path during an optional mixingoperation. In addition, or as an alternative, a gas permeable membranethat prevents the passage of fluid may be placed in the path betweenmacrofluidic centrifugation chamber 200 and port 260 to prevent fluidfrom reaching port 260. This gas-permeable membrane may also beconfigured to serve as a filter to prevent the ingress of airbornemicrobes from the environment or from the air displacement device.Alternatively, the path between port 260 and macrofluidic centrifugationchamber 200 can be designed to possess high fluidic resistance, suchthat under the prevailing conditions, liquid will be prevented fromproceeding all the way to port 260. Likewise an additional valve,gas-permeable membrane or high fluidic resistance conduit may be placedbetween chambers 210 and vent 270 and between chamber 220 and vent 260to prevent the egress of liquid from the cartridge and/or the egress oringress of pathogens and other contaminants via these ports.

During the centrifugation steps, it will typically be important toensure that liquid does not flow between chambers. It is noted that ifports 252 and 257 remain above the surface of the liquid in chambers 200and 220, respectively, during centrifugation, and valves 255 and 250remain open, liquid from the chambers 200 and 210 will fill the channels240 and 230 up to the free surface levels in the respective chambers butliquid will not flow into chambers 220 and 200, respectively.Accordingly, in some example implementations, valves 255 and 250 mayoptionally be omitted from the cartridge unless required for modes offluid transfer described above, or other reasons or modes of operation.

In some example implementations, valves 255 and 250 may be closed duringcentrifugation to prevent the liquid from entering channels 240 and 230respectively. In this case the valves are preferably configured toco-operate with one or more latching mechanisms, such that the valvesremains closed when the cartridge interfacing assembly 130 is notengaged with the cartridge 120. It is noted that it may be preferable toclose one or more of the valves prior to centrifugation, since highfluidic pressures may develop in the distal regions of chambers 200 and210 and channels 240 and 230. For example, pressures in the range of 100psi, 200 psi or 400 psi or greater may occur as a result of highcentrifugal speeds. The chambers can thus be formed such that suchpressures can be withstood. Suitable materials and geometries of thechambers for withstanding such pressures will be known to those skilledin the art. However, some methods for construction of the microfluidicdevice 205 may not be able to sustain these pressures such as, forexample, laminates bonded with pressure sensitive adhesives.Accordingly, and depending on the centrifugal force applied, it may benecessary to locate valves at the opening to the chambers in order toprevent fluids from exiting the chambers and entering the conduitsduring centrifugation. In such cases, it may also be preferable toevacuate liquid from conduits prior to centrifugation.

When latching valves (e.g. valves having an integrated latchingmechanism, or valves configured to be actuated by a latching mechanism)are employed, the cartridge interfacing assembly 130 may be employed toactively and selectively, as required, engage the latching mechanism toopen the valves when the cartridge interfacing assembly 130 isinterfaced with the integrated fluidic processing cartridge, and then toclose and relatch the valves prior to disengagement from the cartridgefor subsequent centrifugal operations. Some valves and associatedlatching mechanisms may be configured to be self-latching, such thatthey latch into a closed position upon disengagement of the cartridgeinterfacing assembly.

Non-limiting examples of suitable latching mechanisms include a ratchetdevice which locks the valve closed and is released by the cartridgeinterfacing assembly to open the valve, or a spring-loaded assemblywhich holds the valve closed by spring force and which is overcome bythe cartridge interfacing assembly to open the valve. Such mechanisms,and other types of known latching mechanisms, may be adapted for thepresent purpose by those skilled in the art. The latching mechanism maybe integrated into the cartridge 120 or may be integrated into acartridge receptacle 112 included as part of centrifuge 110 in FIG. 1.

Sample (e.g. an original sample or a pre-processed sample) may beintroduced into integrated fluidic processing cartridge 120 according tomany different embodiments and methods. In one example embodiment,integrated fluidic processing cartridge 120 may include a removable lidor cap that may be opened to introduce a sample into the cartridge, suchas directly into macrofluidic centrifugation chamber 200, where theremovable cap or lid is sealable (e.g. with an O-ring or other suitablemechanism) in an air-tight manner. Alternatively the lid or cap maycontain a pierceable membrane which allows a needle to penetrate themembrane and deposit the sample into the macrofluidic centrifugationchamber. Such a pierceable membrane should be resealable and capable ofmaintaining a seal to the extent that the pressures required foroptional liquid transfer embodiments described above can be maintainedin the microfluidic centrifugation chamber. Alternatively such apierceable membrane may be provided elsewhere on the cartridge 120 ormicrofluidic device 205 and equipped with a conduit to allow flow to thecentrifugal chamber and optionally a shut off valve to prevent loss offluid or pressure during subsequent operations.

In another example embodiment the sample may be initially provided inanother chamber within integrated fluidic processing cartridge 120, suchas in a sample receiving chamber, and where the sample may becontrollably introduced into the macrofluidic centrifugation chamberaccording to the valving and flow actuation methods described herein. Afurther alternative example embodiment for introducing a sample intointegrated fluidic processing cartridge 120 is illustrated in FIG. 5 anddescribed in further detail below.

Following centrifugal separation and optionally the washing operation,the sedimented sample may be removed in a similar fashion by opening aremovable lid or cap and using a syringe, pipette or other device toaspirate the final sample from the macrofluidic centrifugation chamber.Likewise a pierceable membrane may be provide on the lid to allowremoval of the final sample using a needle and syringe or otheraspiration device.

Macrofluidic centrifugation chamber 200 may be pre-filled with a buffer,diluent, detergent or other specially formulated sample pre-treatmentsolution prior to the introduction of a sample. The sample pre-treatmentliquid may be a solution or buffer that contains one or more componentsor active agents to modify one or more impurities or other components ofthe sample. For example, the sample pre-treatment solution may act onthe sample for the removal, inactivation, digestion, or othermodification of an impurity or other component that may reside withinthe sample. In another embodiment, the required components are includedin the chamber in dried format and these components are dissolved in theliquid sample upon its introduction to the macrofluidic centrifugationchamber.

In other example embodiments, such a pre-treatment liquid may beinitially provided in another chamber within integrated fluidicprocessing cartridge 120, such as in a pre-treatment storage chamber,and where the pre-treatment liquid may be controllably introduced intothe macrofluidic centrifugation chamber according to the valving andflow actuation methods described herein.

In a further embodiment the sample pre-treatment solution may bepre-mixed with the sample prior to the introduction of the sample intothe cartridge. An example of a sample pre-treatment liquid is a bloodlysis liquid, as described in PCT Patent Application No.PCT/CA2013/000992, titled “APPARATUS AND METHOD FOR PRE-TREATMENT OFMICROBIAL SAMPLES”, filed on Nov. 26, 2013, which is incorporated hereinby reference in its entirety.

In yet another example embodiment, a pre-treatment solution may beintroduced into the macrofluidic centrifugation chamber from an externalchamber that is fluidically interfaced to the integrated fluidicprocessing cartridge via a fluidic connector, as described elsewhereherein.

Referring again to FIG. 1, an example implementation of control andprocessing unit 140 is illustrated. Control and processing unit 140 mayinclude one or more processors 145 (for example, a CPU/microprocessor),bus 142, memory 155, which may include random access memory (RAM) and/orread only memory (ROM), one or more internal storage devices 150 (e.g. ahard disk drive, compact disk drive or internal flash memory), a powersupply 180, one more communications interfaces 160, external storage165, a display 170 and various input/output devices and/or interfaces175 (e.g., a receiver, a transmitter, a speaker, a display, an outputport, a user input device, such as a keyboard, a keypad, a mouse, aposition tracked stylus, a position tracked probe, a foot switch, and/ora microphone for capturing speech commands).

Although only one of each component is illustrated in FIG. 1, any numberof each component can be included in the control and processing unit140. For example, a computer typically contains a number of differentdata storage media. Furthermore, although bus 142 is depicted as asingle connection between all of the components, it will be appreciatedthat the bus 142 may represent one or more circuits, devices orcommunication channels which link two or more of the components. Forexample, in personal computers, bus 142 often includes or is amotherboard.

In one embodiment, control and processing unit 140 may be, or include, ageneral purpose computer or any other hardware equivalents. Control andprocessing unit 140 may also be implemented as one or more physicaldevices that are coupled to processor 145 through one of morecommunications channels or interfaces. For example, control andprocessing unit 140 can be implemented using application specificintegrated circuits (ASICs). Alternatively, control and processing unit140 can be implemented as a combination of hardware and software, wherethe software is loaded into the processor from the memory or over anetwork connection.

Control and processing unit 140 may be programmed with a set ofinstructions which when executed in the processor causes the system toperform one or more methods described in the disclosure. Control andprocessing unit 140 may include many more or less components than thoseshown.

While some embodiments have been described in the context of fullyfunctioning computers and computer systems, those skilled in the artwill appreciate that various embodiments are capable of beingdistributed as a program product in a variety of forms and are capableof being applied regardless of the particular type of machine orcomputer readable media used to actually effect the distribution.

A computer readable medium can be used to store software and data whichwhen executed by a data processing system causes the system to performvarious methods. The executable software and data can be stored invarious places including for example ROM, volatile RAM, non-volatilememory and/or cache. Portions of this software and/or data can be storedin any one of these storage devices. In general, a machine readablemedium includes any mechanism that provides (i.e., stores and/ortransmits) information in a form accessible by a machine (e.g., acomputer, network device, personal digital assistant, manufacturingtool, any device with a set of one or more processors, etc.).

Examples of computer-readable media include but are not limited torecordable and non-recordable type media such as volatile andnon-volatile memory devices, read only memory (ROM), random accessmemory (RAM), flash memory devices, floppy and other removable disks,magnetic disk storage media, optical storage media (e.g., compact discs(CDs),digital versatile disks (DVDs), etc.), among others. Theinstructions can be embodied in digital and analog communication linksfor electrical, optical, acoustical or other forms of propagatedsignals, such as carrier waves, infrared signals, digital signals, andthe like.

Some aspects of the present disclosure can be embodied, at least inpart, in software. That is, the techniques can be carried out in acomputer system or other data processing system in response to itsprocessor, such as a microprocessor, executing sequences of instructionscontained in a memory, such as ROM, volatile RAM, non-volatile memory,cache, magnetic and optical disks, or a remote storage device. Further,the instructions can be downloaded into a computing device over a datanetwork in a form of compiled and linked version. Alternatively, thelogic to perform the processes as discussed above could be implementedin additional computer and/or machine readable media, such as discretehardware components as large-scale integrated circuits (LSI's),application-specific integrated circuits (ASIC's), or firmware such aselectrically erasable programmable read-only memory (EEPROM's) andfield-programmable gate arrays (FPGAs).

Referring now to FIG. 3, a flow chart is provided that describes anexample method of performing automated centrifugal separation andwashing of a sample using an integrated fluidic processing cartridgeembodiment shown in FIGS. 2A-C. It will be understood that the presentexample method illustrates but one non-limiting example method, and thata wide variety of other methods may be employed according to theteachings of the present disclosure (for example, methods that do notrequire valves, as described above, or methods that do not requirelatching of the valves during centrifugation). According to the presentexample embodiment, valves 250 and 255 are configured to be latched in aclosed configuration when not engaged by a valve actuation mechanism ofcartridge interfacing assembly 130. Specific example embodimentsdescribing the operation of such valves are described in detail below.

At 300, sample is initially added to macrofluidic centrifugation chamber200, according one of various methods described in the presentdisclosure, such as direct addition through a removable lid or cap inmacrofluidic centrifugation chamber 200, or though extraction from asample chamber that is interfaced with integrated fluidic processingcartridge 120, as described in an example implementation provided below.This operation and subsequent operations not requiring fluid transfer onthe cartridge are optionally performed with valves 250 and 255 in aclosed state to prevent sample from entering conduits 230 and 240. Thisclosed state of the valves may be achieved by controlling cartridgeinterfacing assembly 130 such that the valves are actively actuated in aclosed state or, if the valves are of the latched closed type, theactuation mechanism places the valves in the latched closed state. Afteraddition of the sample to macrofluidic centrifugation chamber 200, thesample and optionally a pretreatment liquid, the latter of which may bepresent or introduced into the macrofluidic centrifugation chamberduring this step, may optionally be mixed, as shown at 305. Such mixingmay be provided by a variety of mechanisms, such as, for example, cyclicor random rotary motion of centrifuge 110, a vibrating mechanism thatmay be built-into the centrifuge, or capable of removable engagementwith centrifuge 110 and/or integrated fluidic processing cartridge 120.This motion may be orbital with an orbital diameter in the range of 1 mmto 10 mm and an orbiting speed in the range of 60 RPM to 2000 RPM forexample. The motion may also be non-circular or linear and may beapplied only at or near one end of the cartridge or cartridge receptaclewhich is otherwise supported on a hinge mechanism at or near theopposite end. The mixing may also be performed by an inversion mechanismfor cyclically inverting or partially inverting the integrated fluidicprocessing cartridge. The mixing mechanism may be integrated incartridge interfacing assembly 130 which is suitably engaged with thecartridge, motorized rotor, or cartridge receptacle included inmotorized rotor to impart a cyclic inversion or partial inversion to theintegrated fluidic cartridge or cartridge receptacle containing thecartridge. The inversion may be such that centrifugation chamber isoriented with its top surface facing downwards and its axis vertical orpositioned at an angle from the vertical axis which is in the range of 0to 90 degrees from the vertical.

Following the optional mixing, centrifugal sedimentation 310 isperformed, whereby integrated fluidic processing cartridge 120 iscentrifuged by centrifuge 110 such that the particulate matter (e.g.cells, such as microbial cells) in macrofluidic centrifugation chamber200 are sedimented. It will be understood that the centrifugation isperformed without engagement of cartridge interfacing assembly 130, suchthat the motorized rotor 114 of the centrifuge 110 may rotate, and suchthat valves 250 and 255 are latched in a closed configuration.

The rotation speed of motorized rotor 114 that is suitable forsedimentation will depend on a number of parameters associated with thesample that is to be centrifuged. For example, suitable parameters forthe centrifugation of microbial cells obtained from a blood sample afterlysis of blood cells with the assistance of a sample treatment solutionare provided in PCT Patent Application No. PCT/CA2013/000992. Knowledgeof the target particulate properties, the suspension fluid solutionproperties and the rotor geometry can be used by those skilled in theart to determine the appropriate speed and time to effect the desiredsedimentation of the particulate. Alternatively the sedimentation speedand time can be determined empirically. In some embodimentssedimentation of all or substantially all of the target particulate inthe liquid is desired and sedimentation parameters are selected toenable all such particles to reach the region of the centrifuge chamberbeyond the supernatant extraction port 256 or optionally to reach thefurthest radial extent in the centrifuge chamber during centrifugation.Alternatively, in the case of samples suspected of containingparticulates with different sedimentation coefficients as a result ofdifference in size or density, it may be desired to retain a portion ofthe particulate having sedimentation coefficients in a desired range andthe sedimentation parameters are selected to enable such a portion ofthe particulate to enter into the region of the centrifuge chamberbeyond the supernatant extraction port 256.

Following centrifugal sedimentation, a portion of the resultingsupernatant is extracted. Prior to extraction of the supernatant,motorized rotor 114 is allowed to come to rest, and cartridgeinterfacing assembly 130 is engaged with integrated fluidic processingcartridge 120 through microfluidic device 205 and valve 255 is opened,as shown at 315. Cartridge interfacing assembly 130 also engages the airdisplacement device with port 260 and actuates the air displacementdevice to produce a positive pressure difference between macrofluidiccentrifugation chamber 200 and vented supernatant chamber 220, resultingin the extraction of supernatant from macrofluidic centrifugationchamber 200 to supernatant chamber 220 as shown at 320. Thus airdisplacement induced flow of the supernatant occurs through supernatantextraction port 256 and supernatant delivery channel 240. The volume ofsupernatant which is thereby removed from the macrofluidiccentrifugation chamber 200 may be controlled, at least approximately, bydisplacing an equivalent volume of air into the macrofluidiccentrifugation chamber by the air displacement device. Alternatively airdisplacement into the macrofluidic centrifugation chamber may beperformed until the supernatant level reaches the supernatant extractionport 256 and no further supernatant can be removed. Generally the volumeof air which must be displaced in this operation can be predeterminedfrom the known liquid volume in the centrifuge chamber. In the lattercase the volume of supernatant removed from the microfluidiccentrifugation chamber, or alternatively the volume of supernatantretained in the macrofluidic centrifugation chamber is determined by thelocation of the supernatant extraction port 256 in the centrifugechamber.

In example embodiments involving washing and resuspension of the washedsediment, some considerations for the location of supernatant extractionport 256 may be the volume of residual supernatant which is requiredafter each wash or after the final particulate resuspension step 342,and the required wash dilution factor discussed in more detail below.Another consideration for a suitable location of supernatant extractionport 256 is one for which the extraction of the supernatant does notdisturb the sedimented particles, for example as a result ofhydrodynamic forces resulting from the flow out of supernatantextraction port 256 which may resuspend all or a portion of thesedimented particulate.

Following the supernatant extraction the sedimented particulate mattermay be resuspended into the residual fluid by a mixing operation asshown at 342 and collected, as shown at 345 without any wash steps.Collection of the resuspended particles in the residual fluid, hereincalled the final particulate suspension, may be done by pipette orsyringe via an openable cap or pierceable membrane on centrifuge chamber200. An alternate embodiment is discussed below where an additionalopening in centrifuge chamber allows the final particulate suspension tobe removed in a similar fashion to the removal of supernatant discussedabove.

In some embodiments a wash operation or a sequence of wash operations isrequired for which a quantity of diluent liquid may be dispensed fromdiluent chamber 210 to macrofluidic centrifugation chamber 200 as shownat 325. Valve 255 is closed and valve 250 is opened, bringingmacrofluidic centrifugation chamber 200 into fluid communication withdiluent chamber 210. Diluent liquid is dispensed into macrofluidiccentrifugation chamber 200 by engaging the air displacement mechanismconnector with port 260, and controllably evacuating air frommacrofluidic centrifugation chamber 200, as shown at 330. Thus airdisplacement induced flow of the diluent liquid occurs through diluentdelivery channel 230. The location of the diluent extraction port 251 atwhich diluent delivery channel 230 enters macrofluidic centrifugationchamber 200 is preferably positioned above the highest extent of theliquid level that is achieved within macrofluidic centrifugationchamber.

Following the dispensing of diluent liquid, cartridge interfacingassembly 130 is optionally engaged as required for the mixing operation,shown at 332, to re-suspend the sedimented particulate matter and mixthe residual supernatant with the diluent liquid in macrofluidiccentrifugation chamber 200.

Following the optional mixing step, the cartridge interfacing assemblyis disengaged as shown at 335 and centrifugal sedimentation is againperformed to re-sediment the particulate material, as shown at 310, andthe cartridge interfacing assembly is re-engaged with the cartridge asdescribed at 315. After having removed the supernatant as at 320, a washcycle is deemed to have been performed. If a single wash cycle isrequired, the sedimented particulate matter may be resuspended into theresidual fluid as shown at 342 and collected, as a concentratedsuspension, as shown at 345. Alternatively, one or more additional washcycles may be performed, by repeating 325-335 and 310-320 one or moretimes. The number of wash cycles required may be determined byperformance requirements which may be related to a required dilutionfactor. The wash cycle dilution factor DF may be calculated from theresidual volume (V_(R)) of supernatant remaining in the centrifugalchamber after step 320 of FIG. 3 and the volume of diluent (V_(D))dispensed into the macrofluidic centrifugation chamber in step 330according to DF=(V_(D)+V_(R))/V_(R).

As noted above, the fluidic paths or conduits between the variouschambers of integrated fluidic processing cartridge 120 are controllablyopened or closed with valves. Although specific examples of valves areshown in many of the examples provided herein, it will be understoodthat valves may employ any suitable mechanism compatible with the fluidpath or port on the device, including, but not limited to pinch valves,ball valves, diaphragm valves, disc valves and plug valves. Examplesimplementations of specific valves are provided below.

In an alternative embodiment, fluid transfer between chambers ofintegrated fluidic processing cartridge 120 may be actuated duringcentrifugation. For example, such an embodiment may be performedemploying centrifugally induced pressure to express the supernatantthrough supernatant extraction port 256 and supernatant delivery channel240 to the supernatant chamber 220. After a sufficient amount of time,during which centrifugation occurs while valve 255 is open, thesupernatant surface, which was initially higher than supernatantextraction port 256, will reach the level of the bottom of supernatantextraction port 256 and supernatant transfer will be complete. The valve255 may then be closed for subsequent process steps.

For this embodiment the supernatant delivery channel 240, supernatantdelivery port 257 and the free surface of liquid in supernatant chamber220 must all have a centrifugal radial position equal to or greater thanthe final centrifugal radius of the free surface of the supernatant inthe macrofluidic centrifugation chamber 200. An example implementationof such an embodiment is illustrated in FIG. 2D, where the supernatantchamber is positioned below the macrofluidic centrifugation chamber 200such that the supernatant deliver port 257A has a centrifugal radius,during centrifugation, that is greater than the supernatant extractionport 256. This embodiment may be beneficial in that the sedimentedparticles will be held firmly by centrifugal force during extraction ofthe supernatant and there is less risk of disturbing the sediment byhydrodynamic forces produced by the exiting supematant flow. This mayallow supernatant extraction port 256 to be placed lower withinmacrofluidic centrifugation chamber 200 than is the case when thesupernatant is removed, as described previously, while the motorizedrotor 114 is at rest. A lower position of the supernatant extractionport 256 will produce a lower residual supernatant volume and a highwash efficiency and a highly concentrated suspension may also beachieved.

In such an embodiment, valve 255 is controllably actuated duringcentrifugation. Such controllable actuation may be achievedelectromagnetically, through the use of electromagnet actuators housedwithin motorized rotor 114 that are externally connected to a controller(e.g. control and processing unit 140, or an electrical controller thatis interfaced with control and processing unit 140), via a rotaryinterfacing mechanism such as a slip ring. In other embodiments, thevalves may be actuated during centrifugation via a pneumatic actuationmechanism residing on centrifuge 110, where the pneumatic actuationmechanism is interfaced with an external pneumatic pressure source via afluid rotary joint.

In another example implementation, the integrated fluidic cartridgereceived within the receptacle with a mechanism that permits theapplication of pressure differential between chambers duringcentrifugation, without requiring the motorized rotor to come to rest.Such an embodiment may be beneficial in reducing overall processingtimes by avoiding the time involved in stopping the motorized rotor andaligning the integrated fluidic cartridge with the cartridge interfacingassembly, and for avoiding the need to align the cartridge interfacingunit with the integrated fluidic cartridge. The motorized rotor may becontrolled to reduce its rotation speed during the application of apressure differential between chambers (and during actuation of valves),in order to reduce centrifugal forces within the channels. It will beunderstood that other non-fluidic components, such as an opticaldetection system, may additionally or alternatively integrated with themotorized rotor.

For example, a pump mechanism may be integrated with the motorized rotoror the receptacle, and wherein the pump is electrically interfaced withan external controller (for example, through an electrical slip ring),such that the pump can be actuated and controlled during rotation of themotorized rotor. The pump should be constructed and oriented towithstand the centrifugal forces during rotation at high speeds.Alternatively, an external air displacement pump mechanism may beemployed that is interfaced with the cartridge via a fluid rotary joint(where the air optionally includes one or more valves).

FIGS. 4A and 4B illustrate an example embodiment in which an additionalfluidic path is provided for transferring the final particle (e.g.cells) suspension to the microfluidic device for further processingfollowing centrifugal separation and washing. In FIG. 4A, a sedimentextraction channel 282 is provided that connects the sediment extractionport 281, which resides in the distal region of the macrofluidiccentrifugation chamber 200 (e.g. at the bottom of the macrofluidiccentrifugation chamber, or at another location associated with thesedimentation of the sediment within the distal region) with themicrofluidic device, and is controlled via sediment extraction controlvalve 280. Sediment extraction channel 282 may lead, for example, to astorage chamber in the microfluidic device for subsequent collection orprocessing or to an exit port designed for collecting the sample by anexternal means.

Alternatively, shown in FIG. 4B, the sediment extraction port 281resides within macrofluidic centrifugation chamber 200 at some heightoffset above the bottom of the macrofluidic centrifugation chamber andbelow the supernatant extraction port 256. This example embodimentallows for the removal of the top portion of the final particlesuspension.

In one example implementation, the sediment may include more than onetype of particle, and a first subset of particles may have a larger sizethan a second subset of particles. In some applications, it may bedesirable to separate (at least a portion of) the second set ofparticles from the first set of particles. The suspension obtained afterthe resuspension step 342 may be centrifuged for a predetermined lengthof time at a predetermined speed such that the first set of particles,having the higher sedimentation rate, move to a position below thesediment extraction port 281, such that a particle suspension free ofthese particles may be removed at sediment extraction port 281.

Additional openings, valves and fluidic conduits may be introducedbetween the distal region of the centrifuge chamber and the supernatantextraction port 256 such that a sequence of extractions through theseopenings from the uppermost to the lowest can be performed to obtain aseries of particle suspensions from each respective level of the finalparticle suspension, optionally allowing the extraction and optionalcollection of fractionated suspensions. Optionally, following the finalparticle resuspension step 342, a controlled centrifugation step may beperformed which the sequence of extractions from uppermost to lowestopenings would yield a series of particle suspensions which containparticles with increasing particle sedimentation rates.

In another embodiment the sediment extraction port 281 may be positionedjust above the meniscus of a cushioning liquid that is configured toretain separated particles, such as microbial cells. This embodiment isdiscussed in more detail below, with reference to FIG. 5.

Although many of the example embodiments described herein employ anintegrated fluidic processing cartridge that includes a supernatantchamber and a dilution chamber, it will be understood that other exampleembodiments, the integrated fluidic processing cartridge may be absentof one or more of such chambers. For example, the integrated fluidiccartridge may include a macrofluidic centrifugation chamber that isinterfaced, through a lateral surface thereof, with the microfluidicdevice, in the absence of the supernatant chamber and the diluentchamber. Such a device may be employed to perform centrifugal separationof a sample, and the extract a sediment into the microfluidic device,optionally for further fluidic processing therein. In another exampleembodiment, the integrated fluidic processing cartridge may include amacrofluidic centrifugation chamber that is fluidically interfaced,through the microfluidic device, to a supernatant chamber, for theseparation of the supernatant from the sediment after centrifugation, inthe absence of a diluent chamber. Such an embodiment may be useful inapplications in which it is the supernatant that is the component ofinterest for further fluidic processing. In such an embodiment, thesupernatant chamber may be fluidically interfaced, through a portprovided therein, to the microfluidic device, for extraction of thesupernatant into the microfluidic device and optional additional fluidicprocessing therein.

In some embodiments, integrated fluidic processing cartridge 120 mayinclude one or more integrated sensors for detecting liquid levels,pressure and/or liquid flow, during operation. Such embodiments may beuseful in verifying system performance as internal process controls. Inone example implementation, one or more electrodes may be placed withinany one or more of the various chambers present within integratedfluidic processing cartridge 120. For example, a plurality of electrodesmay be placed at different locations along the long axis of macrofluidiccentrifugation chamber 200, and the electrodes may be interrogatedrelative to a reference electrode or reference voltage in order todetermine whether or not a given electrode is in contact with liquid,thereby enabling the detection of discrete liquid levels within thechamber. One or more electrodes may be located at locations such as, forexample, above a meniscus level associated with the residual liquid thatis retained after extraction of the supernatant through supernatantextraction port 256. An electrode may also be located adjacent to, orimmediately below, port 260, in order to provide an indication as towhether or not port 260 is contaminated with liquid. An electrode may belocated at a desired level in the macrofluidic centrifugation chamber200 indicating that a sufficient amount of sample and/or diluent ispresent. A reference electrode may be placed sufficiently low in themacrofluidic centrifugation chamber such that the reference electrode isalways submerged in the residual fluid in the macrofluidiccentrifugation chamber and such that the above levels may be detected bycontinuity or resistance measurement between the various electrodes andthe reference electrode.

The sensed electrical signal may be monitored during fluid transfer whenthe cartridge interfacing assembly 130 is engaged with the cartridge 120or receptacle 112. The electrical signal may also be monitored duringcentrifugation according to any one of a variety of transduction methodsand mechanisms, such as, for example, an optical transponder thatrotates with motorized rotor 114 and transmits (and optionally receives)optical signals to (and optionally from) a fixed transponder that doesnot rotate, a pair of wireless transceivers (one of which rotates withmotorized rotor 114), or an electrical connection to control andprocessing unit 140 through an electrical slip ring. Impedancemeasurements may be performed in order to measure or characterize one ormore aspects of the liquid within a given chamber, for example, toverify hemolysis of blood cells within macrofluidic centrifugationchamber 200. Additionally or alternatively, one or more pressure sensorsmay be provided within integrated fluidic processing cartridge 120, inorder to dynamically interrogate the pressure within integrated fluidicprocessing cartridge 120 during rotation of motorized centrifuge.

In other example implementations, liquid level sensing may be achievedusing an external imaging camera that obtains images of the integratedfluidic processing cartridge during rotation (using a camera with asufficiently last frame rate), where the imaging camera is optionallysynchronized to periodically obtain frames when integrated fluidicprocessing cartridge 120 is in a given angular position (optionallyobtaining one image per n rotations, where n>1), thereby enablingdynamic tracking of liquid levels and liquid transport. In order toachieve imaging with sufficient clarity, it may be beneficial totemporarily reduce the rotation rate of the rotor. In other exampleembodiments, liquid levels may be obtained by directing one or morelight beams (e.g. focused or collimated laser beams) onto the cartridge,and monitoring the reflected signal to determine when the beamencounters a liquid within the integrated fluidic processing cartridge.Such a beam may optionally be scanned in order to sample various regionsof the integrated fluidic processing cartridge for liquid leveldetection.

The aforementioned liquid level sensing example embodiments may also beuseful for monitoring the transfer of supernatant or other fluids duringcentrifugation according to the above-mentioned example embodiments, andthe sensed liquid levels may be employed to control the closure of thevalves and/or the application of a pressure differential betweenchambers.

With reference to the example schematic representation in FIG. 5, anexample integrated fluidic processing cartridge 500 is portrayed whichincorporates elements suitable for automated separation and washing ofparticles in a liquid to obtain a concentrated suspension, for example,in accordance with the methods disclosed in PCT Patent Application No.PCT/CA2013/000992. The example integrated fluidic processing cartridgeincludes a sample transfer receptacle 501, a macrofluidic centrifugationchamber 502, a diluent chamber 504 and a supernatant chamber 506.Diluent chamber 504 is prefilled with a wash buffer fluid 505, isfluidically connected to macrofluidic centrifugation chamber 502 viaconduit 510 equipped with shutoff valve 512, contains a vent toatmosphere 515 and is otherwise closed. Supernatant chamber 506 isfluidically connected to macrofluidic centrifugation chamber 502 via aconduit 511 equipped with shutoff valve 513, contains a vent toatmosphere 516 and is otherwise closed. Macrofluidic centrifugationchamber 502 has a conical or round bottom shape and a smooth innersurface which minimizes adsorption or trapping of particles (e.g.microbial cells) during centrifugation and is closed with the exceptionof the openings 522, 523, 524, 525, 526 to respective conduits.

In some example embodiments, macrofluidic centrifugation chamber may beemployed for the processing of blood-containing samples (e.g. wholeblood, blood culture samples, or other blood-containing samples). Insuch embodiments, macrofluidic centrifugation chamber may contain apretreatment fluid 503 which may include agents for lysis of blood cellsand a cushioning fluid 529 to aid in microbial cell recovery and tominimize compaction injury of the cells which may compromise theintegrity and recovery of the target nucleic acids.

The cushioning fluid is of higher density than the remainder of thefluid and is water immiscible such that it settles to the bottom ofmacrofluidic centrifugation chamber under gravity and centrifugalforces. The sample transfer receptacle is equipped with a needle 507which is mounted at the bottom of the receptacle. The needle isconnected to a fluid path 508 equipped with a shut-off valve 509 whichleads to macrofluidic centrifugation chamber 502. A sample tube orcontainer 520 with a pierceable cap 521, such as, for example aVacutainer® blood collection tube or a blood culture tube containing ablood sample and growth media, may be inserted into the sample transferreceptacle such that the needle 507 pierces the cap 521 thus allowingtransfer of a sample fluid to the cartridge via the needle and fluidicpath 508. Optionally needle 507 is covered with a pierceable hood 508which protects the needle from contamination.

The example integrated fluidic processing cartridge 500 is a closedcartridge (apart from the vents described below) which, following theinsertion of the sample, performs all the functions required forseparation and washing of a concentrated suspension within the chambersand conduits of the cartridge, has all reagents and solutions stored inchambers on the cartridge, and retains all excess liquids includingwaste supernatant in chambers on the cartridge. One or more of the ventsand ports may be protected by air permeable membranes with a pore sizesufficiently small to prevent the ingress of microbial pathogens in thetarget range of the device. According to the present example embodiment,all excess and waste liquids are stored on the cartridge and are notexposed to the user. Thus the closed cartridge provides a device whichprotect the user from direct contact with the sample and for which thesample is not susceptible to contamination by external factors duringthe separation and washing process.

As noted above, an automated separation and washing process is generallydescribed in FIG. 3. The cartridge is inserted into an instrumentequipped with the necessary devices and functionality, including acartridge interfacing assembly, as described generally in FIG. 1. Thecartridge interfacing assembly is equipped with all the componentsrequired to perform the necessary actions including actuation of thecartridge valves 509, 512, 513, and 517 and an air displacement devicecapable of application of both positive and negative gauge pressure tothe cartridge centrifuge chamber via cartridge port 518.

The sample tube 520 containing a sample is inserted into the sampletransfer receptacle 501 of cartridge 500 thus piercing the tube cap 521to perform the sample transfer to the macrofluidic centrifugationchamber as shown at 300 of FIG. 3. The cartridge interface assemblyengages with the cartridge via a cartridge receptacle, described indetail below, and is actuated such that valve 509 is open and valves512, 513 and 517 are closed, thus sealing all fluid paths emanating frommacrofluidic centrifugation chamber except the path 508 from the sampletube.

The air displacement device is engaged with the port 518 by way of aconnector which provides a sealed connection with the port. Optionally arigid or flexible tube connects the air displacement device to theconnector. Sample transfer to macrofluidic centrifugation chamber 502 isperformed by operating the air displacement device to extract air frommacrofluidic centrifugation chamber to cause sample flow from the sampletube 520 into macrofluidic centrifugation chamber 502 via fluid path508. The entry 523 of the port 518 must be positioned above the fluidlevel and with a sufficient air gap between the fluid level and theentry 523 such that no fluid flows into entry 523 to the port 518. Theair displacement activated flow is done in a controlled manner such thata predetermined volume of sample is transferred into macrofluidiccentrifugation chamber.

In one embodiment the entry 522 to flow path 508 is also in the air gapabove the fluid level such that, following transfer of the desiredvolume of sample, the air displacement via port 518 can be reversed toprovide a small amount of air displacement into macrofluidiccentrifugation chamber to clear the flow path 508 of sample fluid andmove this residual sample back into the sample tube 520. Then the valve509 is closed and the sample tube 520 is optionally removed from thereceptacle 501.

As noted above, a sample pretreatment fluid may be present in thechamber prior to the sample transfer process or alternatively it may betransferred from a pretreatment fluid tube in a similar manner as thesample. Alternatively a pretreatment fluid storage chamber may beprovided on the cartridge and a fluidic path with valve and an air ventmay be provided to allow the pretreatment fluid to be moved tomacrofluidic centrifugation chamber in a similar manner to the movementof wash buffer to macrofluidic centrifugation chamber as describedbelow.

After addition of the sample to macrofluidic centrifugation chamber 502,the sample and the pretreatment liquid may optionally be mixed as at 305of FIG. 3. A mixing mechanism may be provided whereby the instrumentperforms vortexing, shaking, or cyclic inversion of the cartridge. Thisoperation is done with valves closed on all fluid paths emanating frommacrofluidic centrifugation chamber 502. A valve may be provided on thefluid path to the port 518 to prevent fluid from entering the air pathduring mixing. In addition, or alternatively, an air permeable membranewhich prevents the passage of fluid may be placed in the air pathbetween macrofluidic centrifugation chamber and the port 518 to preventfluid from reaching the port 518. This membrane may also be configuredto serve as an air filter to prevent the ingress of microbes from theenvironment or from the air displacement device. Alternatively the pathbetween the port 518 and the entry opening 523 to macrofluidiccentrifugation chamber can be designed to possess high fluidicresistance such that under the prevailing conditions fluid will beprevented from entering the opening 523 or will be prevented fromproceeding all the way to the port 518. Likewise vents 515 and 516 indiluent chamber 505 and supernatant chamber 506 respectively may beequipped with an air permeable membrane and/or a path with high fluidicresistance to serve a similar purpose.

Following the mixing step 305 a centrifugal sedimentation step 310 isperformed whereby the cartridge interfacing assembly is disengaged fromthe motorized rotor 114 and the cartridge 120 is centrifuged such thatthe particles (e.g. microbial cells) in macrofluidic centrifugationchamber sediment on the cushioning liquid, for example, as per themethods of PCT Patent Application No. PCT/CA2013/000992. The centrifugemay be an angle centrifuge or a hanging bucket centrifuge and thecentrifugal parameters may be selected according to the conditionsprovided in PCT Patent Application No. PCT/CA2013/000992.

The relative centrifugal force applied to the fluids within themacrofluidic centrifugation vessel may be, for example, within the rangeof 1000-15,000 g, or for example, 2,000-12,000 g, or, for example,3000-10,000 g, or, for example, 3000-7,000 g, or, for example,5000-10,000 g, or, for example, 4000-8,000 g. In applications involvingseparation of bacterial and fungal cells from biological samples, it hasbeen found that a suitable relative centrifugal force (RCF) is withinthe range of 1000 g-15000 g range, and more specifically, within therange of 3000 g-7000 g.

Following the centrifugal sedimentation step 310 of FIG. 3, thecentrifuge rotor is stopped and the cartridge interfacing assembly isre-engaged with the motorized rotor as at 315 and extraction of thesupernatant 527 from macrofluidic centrifugation chamber to thesupernatant chamber 506 is performed as at 320 whereby the residual 528containing the target sediment (e.g. microbial cells) is retained at thebottom of macrofluidic centrifugation chamber 502. This action isperformed by opening valve 513 while valves 509, 512 and 517 remainclosed and engaging the air displacement device connector with port 518and controllably displacing air into macrofluidic centrifugationchamber. Thus air displacement induced flow of the supernatant occursthrough fluid path 511, the entry 524 of which is placed below thelowest extent of the supernatant. Optionally the entry 524 is placed atthe lowest extent of the supernatant which is to be expressed frommacrofluidic centrifugation chamber, thus preventing residual 528 frombeing extracted from macrofluidic centrifugation chamber.

Following the supernatant extraction step 320, the wash bufferdispensing steps 325 and 330 are performed whereby wash buffer isdispensed into macrofluidic centrifugation chamber 502. This action isperformed by opening valve 512 while holding valves 509, 513 and 517closed and engaging the air displacement device connector with port 518and controllably evacuating air from macrofluidic centrifugation chamber502. Thus air displacement induced flow of the wash buffer occursthrough fluid path 510. The entry 525 of wash buffer path 510 ispreferably placed above the highest extent of the fluid level inmacrofluidic centrifugation chamber.

Following the wash buffer dispensing step 544, the mixing step 332 isperformed to thoroughly mix the wash buffer and the residual fluid inmacrofluidic centrifugation chamber. This may be performed by vortexing,shaking, or cyclic inversion of the cartridge as described previously.

Following the mixing step 332, the centrifugal sedimentation step 310 isperformed to re-sediment the collected sediment (e.g. microbial cells)and the supernatant is removed from the centrifugal chamber as in step320.

The sequence of steps 325-335 and 310-320 collectively form a wash cyclewhereby the cell suspension is diluted in wash buffer, the particles arere-sedimented and the supernatant is extracted. The wash cycle may berepeated multiple times to effect multiple additional wash cycles asrequired to obtain a final suspension sufficiently dilute (e.g. amicrobial cell suspension that is sufficiently dilute of contaminantsand interferants). The desired dilution factor depends on the samplecomposition and downstream detection procedure. In one embodiment,intended for applications involving separation of bacterial and fungalcells from biological samples, electrical lysis of microbial cells anddetection through RT-PCR amplification of ribosomal RNA, the dilutionfactor is selected in 100-100000 range. More preferred range is1000-50000. In another embodiment involving separation of bacterial andfungal cells from blood samples, lysis of microbial cells and detectionthrough PCR amplification of DNA, the dilution factor can be as small as1 provided that inhibitor-resistant polymerase enzyme along with anappropriate amplicon detection scheme is employed. Exemplaryimplementation of DNA amplification and detection method in whole bloodis reported in prior art (e.g., L. A. Neely et al., Sciencetranslational medicine5.182 (2013): 182ra54-182ra54.).

Following the final supernatant extraction step 320 the mixing step 342is performed to resuspend the sedimented particles (e.g. microbialcells) in the final residual fluid 528 to produce the final suspension.

Following the resuspension step 342 the final suspension is extracted byair displacement through fluid path 510. The volume of the finalsuspension depends on the nature of the application. For instance, whenthe intended application is the detection of pathogenic microbial cellsin whole or cultured blood, the volume of the final cell suspension maybe selected to be in 10 μL-500 μL range. More preferred range is 20μL-120 μL, or 50-100 μL. During the extraction of the final cellsuspension valve 517 is open and valves 509, 512 and 513 are closed andair is displaced through port 518 into macrofluidic centrifugationchamber to displace the fluid out of opening 526 via fluid path 516. Theopening 526 is so positioned at the top surface of the cushioning fluid529 that the final suspension in its entirety, or substantially all ofthe suspension, is expressed from macrofluidic centrifugation chamberwithout expressing any of the cushioning fluid 529 as depicted in FIG.5. Alternatively, the opening 526 is so positioned that the finalsuspension and portion of or all of the cushioning fluid may beexpressed from the macrofluidic centrifugation chamber through fluidpath 516. Fluid path 516 leads to the next downstream cartridge elementwhich in some embodiments may be a chamber or chamber configured toallow retrieval of the final suspension from the cartridge for furtherprocessing outside of the cartridge, and in other embodiments this maybe a fluid path to a suspension collection chamber, or for example, anelectrical lysis chamber as described below.

Integration of Centrifugation-Based Integrated Fluidic ProcessingCartridge with Additional Fluidic Processing Elements

As described below, in various example embodiments of the presentdisclosure, the microfluidic device of integrated fluidic processingcartridge 120 can be supplemented with various additional fluidiccomponents, chambers, and features in order to support furtherprocessing of the final residual suspension (or the supernatant, ifdesired).

In one example embodiment in which cells are present in the finalresidual suspension, after having extracted the supernatant, areresuspended. Then, the cell content of the resulting cell suspension maybe transferred to the microfluidic device, as described above, throughthe sediment extraction port. The cell suspension may then beinterrogated according to any of a wide range of cell assays. In oneexample embodiment, the resulting cell suspension may be delivered to aplanar channel or chamber formed at least in part by a transparentoptical window. Cells retained in the planar channel or chamber may beoptically interrogated.

For example, the retained cells may be enumerated and/or inspected anoptical imaging system equipped with a microscopic objective. Theobjective may be mounted on moving mechanism to scan the volume of thechamber.

In one example embodiment, the cells are located in a zone which islocated in the field of view of the microscope objective. For example,the cells may be retained on a planar substrate coated with a materialsuitable for adhering cells, such as a cell-specific or cell-genericcoating. The cells may also be driven to a focal zone via electricfields, such as via dielectrophoresis.

In order to enable interrogation of low cell counts, the cells may beretained on the surface of a filter housed within the microfluidicdevice. This eliminates or relaxes the requirement for scanning alongthe axis of the objective. An example embodiment in which the cells areretained on filter for microscopic inspection is presented in FIG. 6. Afilter 61, for example a membrane filter, having a thickness less thanthat of the chamber, is secured within a channel of the microfluidicdevice (where the channel is in fluid communication with the sedimentextraction channel for delivery of the concentration suspensionthereto), such that the channel is divided into two portions 62 and 63,thereby enabling cells within the sample to be retained by the filter asthe concentrated suspension is flowed between the inlet port 64 andoutlet port 65. In one example implementation, the filter may be made ofmaterial, such as high density polyethylene, or polycarbonate membrane.The upper part of the channel, 66 in FIG. 6, is made of thin transparentfilms to allow passage of light to the objective.

The microscopic examination of microbial cells, as explained above, maybe used for performing antibiotic susceptibility testing (AST),particularly in the case of non-enriched samples for which the microbialcell count in the sample is low. According to one exampleimplementation, the biological sample is first tested for the presenceand identity of the pathogenic microbial cells using the methodsdescribed in the present disclosure or any other suitable method. Thisdetermination narrows down the selection of appropriate antibiotic agentto one or few candidates, often by referring to the antibiogram of theassociated healthcare setting.

The AST is initiated by incubating two aliquots of the sample bothsupplemented with appropriate medium that sufficiently supports thegrowth of the microbial cells under suitable temperature conditionsprovided by an incubation instrument. The antibiotic agent is added toone of the aliquots and the other aliquot is treated as control sample.After the passage of a predetermined incubation period, the two aliquotsare processed within the microfluidic device portion. Thus relativelyclean cell suspensions are prepared for each aliquot. Then the cells aremicroscopically inspected by retaining in filtered chambers as describedabove to verify if the cells exposed to the antibiotic agent have beenkilled (the case of cidal antimicrobial agents) or have been inhibitedin terms of growth (the case of static antimicrobial agents). Thereby,the AST result is determined. Accordingly, the present exampleembodiments may enables the extending the methods of AST recited in USPatent Application Publication No. 2013/0217063 to the case of sampleshaving scarce microbial count (for example, in the range of 1 to 100,000CFU/ml). It will be understood that the aliquots may be sample aliquotsthat are processed on separate integrated cartridges, or the aliquotsmay be aliquots of the concentrated suspension that is obtained afterautomated centrifugation, thus permitting the aliquots to be split andsubsequently processed within the microfluidic device portion of asingle integrated fluidic cartridge.

In some embodiments, the amount of dilution achieved during fluidicprocessing, prior to delivering the concentrated suspension to themicrofluidic device, may be selected to be sufficiently high such thatthe suspended cells can be retained on the filter without causing filerclogging. A suitable dilution level (or washing level) may be determinedbased on the composition of the biological sample, the nature ofpretreatment of the sample prior to dilution, and the area of thefilter.

This may be illustrated, for example, by referring to a specific examplewhere the target microbial cells are in whole blood. For instance, USPatent Application 2013/0171615 teaches lysing blood cells using equalvolume of 1M NaCarbonate pH 10.0+1% Triton X-100. According to thepresented data 2.5 mL of treated blood can be passed through a membranefilter with diameter of 2.5 cm and pore sizes of 0.45 μm withoutsignificant clogging. Accordingly, only 2.5×(0.4/25)² mL=0.6 μL ofunwashed lysed blood sample can be passed through a filter having adiameter Of 0.4 mm, which approximately corresponds to the field of viewof a 40× microscopic objective. However, a washing procedure providing adilution of blood debris by 100× will enable filtering of 60 μL cellsuspension from pretreatment step. The cell content of the suspensionmay undergo additional fluidic processing prior to microscopicinspection. These additional processing steps may include, for example,exposure to drugs or other chemical agents for a predetermined period,staining with fluorescent dyes, or incubation with appropriate FISH(Fluorescence in situ hybridization) reagents, and addition of cellgrowth media and optional incubation therein. The manipulation can beperformed prior to filtering and after retaining the cells on thefilter.

In an alternative example implementation, the concentrated cellsuspension, extracted to the microfluidic device, and optionallyfiltered therein as described above, may be mixed with a matrix assistedlaser desorption/ionization (MALDI) matrix material and subsequentlyfluidically delivered to a chamber from which a MALDI sample may beextracted for performing MALDI analysis. In one example embodiment, themicrofluidic device may be configured to deliver the mixture to a one ormore wells that are formed on a substrate suitable for MALDI (e.g. ametal substrate) such that the microfluidic device provides one or moreMALDI-ready samples. The MALDI substrate may then be removed from themicrofluidic device and processed according to known MALDI methods.Alternatively, the wells formed on the MALDI substrate may open wells,or may be exposed by removing of one or more peelable or otherwiseremovable layers of the microfluidic device.

The non-limiting example embodiments described below pertain to anexample integrated fluidic processing cartridge in which themicrofluidic device includes components for the lysis of microbial cellsextracted according to the aforementioned embodiments, and assaychambers for performing molecular detection of nucleic acids present inthe lysate.

It will be understood that although many of the example embodimentsprovided herein relate to the purification and concentration of cells ina suspension, the methods, systems, and devices described herein may beadapted to a wide variety of associated embodiments. For example, insome example implementations, the supernatant can be extracted andtransferred to the microfluidic device for further fluidic processing,such as the performing of one or more integrated assays. Such anembodiment would not involve a washing step. In other embodiments, boththe supernatant, and a residual sample may be obtained, and one or bothmay be transferred to the microfluidic device for further processing. Inother embodiments, a fluid, such as a suspension, that is initiallytransferred to the microfluidic device for processing, may besubsequently transferred back to the macrofluidic centrifugation chamberfor further centrifugation.

In the embodiments described a sample, such as a whole blood sample, isinserted into a cartridge and a series of operations are performed onthe cartridge by a dedicated instrument to perform the functionssummarized in FIG. 7A-D.

Thus, as depicted in FIG. 7A, the sample, containing target cells ofinterest, undergoes the automated separation and washing process 530followed by electrical lysis and treatment 531 and then reversetranscription 532 of rRNA which is followed by PCR amplification 533 ofthe cDNA (and/or optionally gDNA) and multiplexed detection 534 of thetarget amplified nucleic acids. The instrument then analyses thedetected signals and reports the results to the user 537. Pretreatmentof a sample is performed by initial selective lysis of non-microbialcells (such as blood cells) and subsequent centrifugal separation andoptional wash cycles, as presented generally in FIG. 3, to concentratethe cells and remove the blood debris. The microbial cells aresubsequently resuspended and the resulting microbial cell suspension 535is herein termed the “final cell suspension”. The final cell suspensionis passed to an electrical lysis chamber where the microbial cells arelysed such that target nucleic acids are released and electricallytreated. The resulting microbial cell lysate 536 is then passed to athermal chamber or a plurality of thermal chambers where reversetranscription, PCR and detection of PCR products is performed asrequired for detection of target microbes. The cartridge provided insome embodiments described herein integrates the totality of thisprocess where a sample, such as a whole blood sample, is introduced tothe cartridge and all elements required for sample pretreatment,centrifugal separation and washing, microbial cell lysis, reversetranscription, PCR and detection of target PCR products are present inthe cartridge which, in conjunction with a dedicated instrument,performs the full process culminating in the detection and optionallythe identification of the target microbes.

Alternative example embodiments may integrate a part of this process.For example, a cartridge incorporating all elements required for thepre-treatment and centrifugal separation and washing process 530 resultsin a final cell suspension 535 which can be retrieved from the cartridgeand processed externally from the cartridge as in FIG. 7B.

In another example embodiment, the cartridge integrates the samplepre-treatment, separation and washing process 530 and microbial cellelectrical lysis and treatment 531, yielding a lysate solution 536 whichcan be retrieved from the cartridge and processed externally as shown inFIG. 7C.

As noted above, integrated fluidic processing cartridge 120 isinsertable into a receptacle supported by a motorized rotor appropriatefor centrifugation, and the integrated fluidic processing cartridge mayincorporate one or more fluidic features (e.g. fluidic valves) such asvalves for opening and closing ports and fluid paths, vents, and portsto allow connection to an air displacement device for air displacementinduced fluidic movements. Valves may employ any suitable mechanismcompatible with the fluid path or port on the device, including, but notlimited to punch valves, ball valves, diaphragm valves, disc valves andplug valves. Valves may be employed to control and/or direct fluidicmovements, to control evaporation of fluids during electrical lysing andtreatment and/or PCR cycling, and to allow superheating to occur in theelectrical lysing and treatment chamber as described in United StatesPatent Application Publication No. 2014/0004501.

Although the preceding example embodiments relate to the processing ofwhole blood as a sample matrix, it is to be understood that the methodsand devices disclosed herein may be adapted to a wide variety ofspecimens. Suitable specimens include, but are not limited to urine,sputum, cerebral spinal fluid, swabbed tissue samples, vaginal samples,and other sample types of biological origin, and non-biological samplesthat may contain microbial cells. A sample may be provided by processinga solid or partially solid sample in order to produce a liquid sample(e.g. using a process such as homogenization). Examples of other sampletypes include other liquid samples that may contain microbial cells,such as environmental water samples, liquid food samples, andhomogenized food samples. The initial sample may be combined with areagent, buffer, or other medium prior to introduction into theintegrated fluidic processing cartridge.

Furthermore, although the preceding example embodiments relate to theamplification and detection of nucleic acids, it is to be understoodthat the methods and devices disclosed herein may be adapted to otherapplications and assays. For example, the lysate may be used to detectcellular proteins, or the lysate may be retrieved from the cartridge forsuch purposes.

Additionally, treatment of the lysate may be performed to prepare thelysate for other applications such as, for example, MALDI TOF massspectroscopy for the phenotypic identification of microbes. Suchapplications may require the integration of a protein solubilizationstep with its required cartridge elements as shown in FIG. 7D. In anexample of such a protein solubilization step, the lysate is passed intoa chamber containing an organic solvent, such as acetonitrile, todissolve as many proteins as possible. Then the cartridge is optionallycentrifuged to sediment cell wall fragments and the supernatant ispassed to a chamber which allows the protein solution to be retrievedfrom the cartridge by the user.

With reference to the schematic representation in FIG. 8 and FIG. 5,some embodiments of the cartridge contain elements provided for theelectrical lysis and treatment operation including a cell suspensionchamber 560 connected via fluid path 516 and opening 519 to macrofluidiccentrifugation chamber 502, electrical lysis chamber 561 and lysatechamber 562. The fluid path between the chambers 560 and 561 and between561 and 562 contain shut-off valve 565 and 566 respectively. To effectfluid flow through this path by way of air displacement via port 518,valves 509, 512, and 513 are closed and valves 517, 565 and 566 areopen. Furthermore an air path and vent must be provided at the furthestdownstream extent of lysate chamber 562 to allow the lysate to flow intothe chamber. The widths and the heights of the cell suspension,electrical lysis, and lysate chambers may be, respectively, selected in1 mm-30 mm and 0.025 mm-1 mm ranges.

The final cell suspension is passed to the cell suspension chamber 560by the extraction step 345 described previously where it is held priorto initiation of the electrical lysis and treatment process.Alternatively the cartridge does not contain a cell suspension chamberand the pretreated cell suspension may be passed directly to theelectrical lysis chamber 561 in the mariner described below. During thisextraction step the downstream valves 565, 566,567 and 572 are open toallow the fluid to flow through the channel 516 and into the holdingchamber 560. Upon completion of the extraction step 345, the cartridgeinterfacing assembly 120 performs the operations necessary for theelectrical lysis operation. The air displacement device attached to port518 is used to displace a portion of the final cell suspension into theelectrical lysis chamber 561 to fill the chamber. The valves 565 and 566are then closed and an electrical pulse train is applied across theelectrodes of the electrical lysis chamber in the manner described in USPatent Application Publication No. US20140004501, titled “METHODS ANDDEVICES FOR ELECTRICAL SAMPLE PREPARATION”, and filed on Jan. 25, 2013,which is herein incorporated by reference in its entirety, to effectmicrobial cell lysis and treatment of the cell suspension in theelectrical lysis chamber producing microbial cell lysate 536.

In one example embodiment, which is intended for detecting pathogenicmicroorganisms in blood samples, the washing fluid is selected to havean ionic strength in 0.1-1 mM range, which is appropriate for thesatisfactory operation of electrical lysis within the required lysisefficiency. The voltage pulse train consists of approximately 300bipolar square pulses at a frequency of 10 kHz and equal amplitudes suchthat the electric field in the chamber is about 10 kV/cm. The cellsuspension is briefly superheated to temperatures above approximately120° C. to effectively lyse fungal cells. To avoid over-pressurizing theelectrical chamber, the electrical chamber temperature is monitoredduring the pulse train to avoid over-pressurizing the chamber. This isdone by monitoring the temperature dependent electrical current passingacross the chamber in accordance with the methods of US PatentApplication Publication No. US20140004501. In one embodiment this isachieved by measuring the peak electrical current averaged over about 5first cycles of the pulse train and setting the maximum allowable peakcurrent at about 3 times of this initial current. When the peak currentreached the maximum allowable value, a control system lowers the pulseamplitude to about ⅓ of its initial value.

Upon completion of the electrical pulse train the valves 565 and 566 areopened and a further volume of pretreated cell suspension is displacedinto the electrical lysis chamber in the same manner thus displacing anequal volume of the microbial cell lysate into the lysate chamber 562via fluid path 568. The volume so displaced may be equal to the fullelectrical chamber volume or optionally a portion of the electricalchamber volume, the former displacing the entire volume of the microbialcell lysate and the latter displacing a portion of the microbial celllysate into the fluid path 568 and chamber 562. The valves 565 and 566are again closed and an electrical pulse train is applied to theelectrical chamber. Further volumes of pretreated cell suspension aresimilarly displaced into the electrical lysis chamber and subjected toelectrical lysis and subsequently displaced into chamber 562. Uponelectrical lysis of the full volume of the cell suspension, oralternatively a portion thereof, the remainder of the microbial celllysate is passed into the lysate chamber 562 by air displacement asdescribed previously. The fluid path 569 emanating from lysate chamber562 may terminate at a port for retrieval of the lysate sample as inFIG. 7C, or may lead to further conduits, valves and chambers requiredfor further processing.

It will be understood that the electrical lysis method described hereinis merely an example of a lysis method, and that other lysis methods maybe used in alternative, such as bead beating, ultrasonic lysis(optionally with bead beating, and chemical lysis).

Reverse Transcription, PCR and Multiplexed Detection

With reference to the schematic representation in FIG. 8, someembodiments of the cartridge contain elements provided for reversetranscription of rRNA into cDNA and PCR amplification and detection ofamplified cDNA and/or gDNA products. Some embodiments intended only forgDNA detection do not contain the elements required for reversetranscription. These elements include a fluid path 569 from the lysatechamber 562 to the thermal chamber or array of thermal chambers 563, apath 570 from the thermal chamber or array of thermal chambers to an airvent 571, optionally a valve 567 in the fluid path 569 , and optionallya valve 572 in the path 570 . Preferably the thermal chamber or array ofthermal chambers contain the required reverse transcription reagents,PCR reagents, and primers in a dry form which respectively contain allconstituents necessary for the reverse transcription and PCR processes.In one embodiment, a master mix solution containing the reversetranscription and DNA polymerase enzymes and appropriate preservatives,is dispensed in dry form on the wall of the thermal chambers. The mastermix solution, containing the reverse transcription and DNA polymeraseenzymes and appropriate preservatives, is dispensed in dry form on thewall of the thermal chambers.

The reverse and forward primers which are generally specific to thetarget microbial cells designated for each thermal chamber, are alsodeposited in dry form on the wall of the thermal chambers. In anotherembodiment the lysate chamber may contain some of these reagents in dryform. In one example embodiment the master mix solution may be depositedin dry form on the wall of lysate chamber 562. In one embodiment, thedrying of master mix solution can be achieved by freeze-drying on thechamber surface. Alternatively, the master mix may be dried in the formof lyophilized beads and stored in the chamber. In another embodimentthe master mix is supplemented with appropriate stabilizer agents beforebeing air or vacuum dried on the surface. Exemplary implantation of thisdrying method has been provided in U.S. Pat. No. 8,900,856.

Upon exposure to the lysate solution the reagents are formulated todissolve readily, aided in some embodiments by fluid flow over the dryreagents, agitation of the lysate fluid in contact with the dryreagents, heating of the fluid chambers which contain the dry reagents,or some combination of these mechanisms. In another embodiment liquidreagents may be stored in neighbouring chambers in the cartridge andfluid pathways and flow control elements are provided to transfer ofsuch liquid reagents into the lysate chamber, the thermal chambers orinto the fluid path to combine with the lysate.

Example Thermal Chamber

In some embodiments the thermal chamber is constructed as shown in FIGS.9A-E. The heights and the diameters of these chambers may be selected tobe, respectively, in 0.025 mm-3 mm and 0.1-5 mm ranges. FIG. 9A shows across-section view of an embodiment of a thermal chamber 580 where 583is a top cover layer or film, 584 is a layer forming the sides of thechamber and 585 is a bottom layer. The chamber in plan view may becircular, as depicted in FIG. 9C, or may alternatively be square,rectangular or multisided. Top layer 583 of chamber 580 is constructedof a transparent material suitable for optical transmission of thewavelengths necessary for fluorescence excitation and measurement of thefluorescence signals from amplified PCR products. Alternatively thebottom layer may be constructed of such materials for this purpose. Inthis way PCR amplification products can be monitored in real time ordetected at appropriate intervals in the thermal cycling process. Thepaths 581 and 582 are provided for fluid flow into or out of the chamberas required. These may be of the full height of the side wall layer 584or, as is depicted in FIG. 9A, one or both of these may be a portion ofthe height of layer 584.

In another embodiment the outflow path 587 is formed in the layeradjacent to the bottom layer 586 as shown in FIG. 9B and the bottomlayer 586 is an air permeable membrane which resists the passage offluid at the working pressure of the cartridge. Such a construction canbe used to eliminate air from the chamber during fluid filling orminimize the occurrence of air bubbles. When more than one thermalchamber is provided, such as for the case of an array of chambers 563,the individual chamber inlets may be fluidically connected to the fluidpath 569 via a network of paths, bifurcations and interconnections.

Alternatively the fluid path 569 may lead to a chamber 595 above thearray of thermal chambers 596, as depicted in cross section view in FIG.9D and in plan view in FIG. 9E. A bottom cavity 598 may also be providedas an alternative to multiple networked paths to connect to path 570. Inthis case the bottom of the thermal chamber may be an air permeablelayer or membrane which prevents the movement of fluid into the cavity598 and path 570.

A heating element 590, shown in FIG. 9C, is provided at the top surface,the bottom surface or the side surfaces of the chamber or somecombination thereof. Heating element 590 may be a resistive heatingelement such as a wire, ribbon, or strip which produces heat by Jouleheating when electrical current is supplied. Non-limiting examplematerials are Nichrome, Kanthal, carbon, copper or platinum.Alternatively, the heater may be formed from an etched metal foil, thinfilm or printed film. Such heating elements may form the bottom layer,top layer or side layer of the chamber or may be placed on or adjacentto one or more of these layers.

In some embodiments, a material or configuration with a high ormoderately high thermal coefficient of resistance is used to formheating element 590, so that the heater temperature can be monitoredallowing some embodiments to employ active feedback control of theheater temperature.

In other embodiments the heating element may be external to integratedfluidic processing cartridge 120. Examples of external heaters includeresistive heater, radiative heater, convection heater, induction heater,or Peltier heater.

To enable thermal cycling, an active or passive cooling mechanisms maybe introduced. Cooling methods include, but are not limited to, externalpassive cooling by heat sinking or active cooling using thermoelectric(Peltier) coolers, air or other fluid convection. Some embodimentspossess integral passive cooling in which the materials of the walls,top and/or bottom layers or layers adjacent to one or more of thechamber surfaces possess thermal properties which allow heat to berapidly conducted away from the chamber and absorbed by the neighbouringmaterials when heating is removed and the chamber temperature is greaterthan the temperature of the heat sinking materials. This may be aided byproviding an external heat sink with a high heat capacity or an externalheat sink which is actively cooled.

To move lysate into the thermal chamber or array of thermal chambers563, the air displacement device connected to port 518 of FIG. 5 may beused to displace air into macrofluidic centrifugation chamber withvalves 509, 512, and 513 closed and valves 517, 565, 566, 567 and 572open, thereby displacing lysate from the lysate chamber to the thermalchambers. In an alternate embodiment the air vent 571 may be also beconfigured as a port which allows connection of an air displacementdevice such as a syringe pump, peristaltic pump, bellows pump or anyother air displacement device or pressure source which can controllablydeliver or remove air. The air displacement device is engaged with theport 571 by way of a connector on the cartridge interfacing assembly 130which provides a sealed connection with the port. Optionally a rigid orflexible tube connects the air displacement device to the connector toallow the air displacement device to be remote from the cartridgeinterfacing assembly. This embodiment allows the liquids in the chambersand conduits of FIG. 8 to be moved in the direction of port 571 byevacuating air via port 571 The valves 517, 565, 566, 567 and 572 in thepath from macrofluidic centrifugation chamber 502 must be open andmacrofluidic centrifugation chamber must be vented to atmosphere via oneof the available paths. Alternatively, an air vent or multiple airvents, controlled by shutoff valves, may be supplied at variouspositions along the fluid path to allow air evacuation from port 571 totransfer fluid. This method of fluid movement may be optionally beapplied to one or more of the following fluid transfer actions: theextraction of pretreated cell suspension from macrofluidiccentrifugation chamber, the transfer of cell suspension into theelectrical lysis chamber, the transfer of lysate into the lysate chamberand the transfer of lysate to the thermal chamber.

Performing RT-PCR in Thermal Chambers

In the embodiments described above for which the master mix includingreverse transcription reagents and required primers are provided in dryform in the lysate chamber and the reverse transcription step may beperformed in that chamber. Thereby. following dissolution of the dryreagent in the lysate solution, the lysate chamber is heated in a mannerand with embodiments similar to that described above for the thermalchambers in accordance with the reverse transcription protocol.Following reverse transcription the solution containing the reversetranscription products (cDNA) is transferred to the thermal chambersalong with the PCR components of the master mix. Forward primers, storedin dry form in each thermal chamber, are released into the liquid mediaand thermal cycling is performed in accordance with a predeterminedsequence of temperatures and dwell times.

Alternatively, dry reagent is dissolved in the lysate solution withinthe lysate chamber and is directly introduced into the thermal chambers.The locally dried reverse and forward primers are thereby released intothe lysate solution and reverse transcription and PCR amplification areperformed.

Optionally, the port 571 is used to apply a vacuum to the thermalchambers to evacuate air from the thermal chambers and minimize thetrapping of air bubbles in the chambers when liquid is drawn into thechambers. Prior to initiation of the PCR the valves 567 and 572 may beclosed to prevent fluid movement and/or expansion of residual airpresent in the thermal chambers during thermal cycling. Optionally,prior to thermal cycling, the thermal chambers may be placed underpressure by closing valve 567 and applying positive pressure to port571. Positive pressure may continue to be applied to port 571 during thethermal cycling process, or alternatively in embodiments containingvalve 572, the valve may be closed after positive pressure at port 571has been applied and prior to thermal cycling. Applying positivepressure will increase the vapour pressure in the thermal chamber andinhibit the creation and growth of air bubbles during the elevatedtemperature portions of the thermal cycles. In alternative embodiments,the pressure may be applied by air displacement via port 518 of themacrofluidic centrifugation chamber.

The amplification of target DNA molecules in the thermal chamber may bemonitored by an optical system. In one example implementation, a lightsource such as an LED may be employed that emits in the wavelength rangecorresponding to the excitation band of the dye used in the PCR mastermix and having no or very little emission in the wavelengths extendinginto the fluorescence emission spectra of the dye. The light from theLED after passing through a wavelength-selective mirror, illuminates theamplicons in the thermal chambers. The fluorescent dye included in thethermal chamber emits in a characteristic spectrum with intensitydependent on the chamber temperature. The emission light after beingreflected from the wavelength selective mirror is imaged on a detectorarray. The wavelength selective mirror significantly attenuates thecontribution of scattered excitation light in the emission beam. Theimaging of the thermal chamber array is performed during a pre-selectedperiod in the temperature cycling procedure of the PCR reaction.Optionally, at the end of thermal cycling the temperature of the thermalarray is scanned with an appropriate rate and the fluorescence signalfrom the chambers is recorded at selected time intervals. This processis intended for performing melting analysis on the amplicons.

An example implementation of the optical system is presented in FIG. 19.The system includes an LED, 410, whose light is collected andsubstantially collimated by the lens combination, 411 and is filtered bypassing through a low pass filter 412 to attenuate the part of spectrumoverlapping with the emission spectrum of the fluorescent dye. Thecollimated beam after reflection from a dichroic mirror 413 and passingthrough microscopic objective 414 illuminates the thermal chamber array745. The objective magnification may be selected according to the sizeof the thermal chamber array 745. For instance, if the thermal chambercovers a spatial dimension of 15 mm×15 mm then a standard microscopeobjective having a magnification in the range of 1×-1.5× may beselected. The fluorescence emission, emanating from the thermalchambers, is collected by the said objective, and after undergoingfiltering by the dichroic mirror 413 is further filtered by the emissionfilter 415. This filtering action further attenuates the signalsoriginating from the excitation source and pass through most of thefluorescence signal from the thermal chamber. The light transmittedthrough the excitation filter is imaged by the lens combination 416 ontoan array of photodetectors 417 which may be in the form of CCD or CMOSsensors.

Although many of the examples provided herein relate to performingRT-PCR on a lysate obtained though performing lysis in the microfluidicdevice, it will be understood that other assays may be performed, suchas PCR of DNA present in the lysate, such as nested PCR. Furthermore, itwill be understood that other detection modalities other than opticaldetection may be employed, such as electrochemical sensing, and sensingvia nuclear magnetic resonance assays known in the art.

Example Integrated Fluidic Processing Cartridge with IntegratedMolecular Assay Microfluidic Device

FIG. 10A shows an example integrated cartridge 700 for microbialidentification in a whole blood sample, which incorporates samplewithdrawal from a Vacutainer type blood sample tube, samplepretreatment, centrifugal separation and washing, electrical lysis andtreatment, reverse transcription, PCR and detection of target PCRamplified products.

Example integrated cartridge 700 is shown having three components, thefirst component 698 comprising the sample transfer receptacle 702,macrofluidic centrifugation chamber 703, the diluent chamber 704 andsupernatant chamber 705. First component 698 may be a single plasticmolded part fabricated from materials which are compatible with the formand function of the device. Alternatively, first component 698 may be anassembly of subcomponents which are plastic parts, molded or formed by ameans consistent with the material, form and function of the device. Inthis respect, the material should be selected to be of sufficiently highstrength to withstand the high centrifugal forces that the cartridgewill be subjected to, and the materials should be compatible with thefluids used and, in the case of molecular applications, should notintroduce contaminants into the pretreated cell suspension which willinterfere with downstream process. Non-limiting examples of materialsfrom which first component 698 can be fabricated are polypropylene,polycarbonate, polyethylene, PET, polystyrene, Cyclic Olefin Copolymeror some variant of these materials.

The second component 699 is a microfluidic device mounted on the lateralface of component 698 comprises fluidic paths and valves connecting thechambers in component 698 and components for electrical lysis, reversetranscription and PCR. The second component 699 is a laminate comprisedof a number of layers in which are formed holes, channels and chambersand electrical components for electrical lysis and heating operations.

The layers may be machined, punched, embossed or molded to form thenecessary features. Each layer may be comprised of either a single ormultiple sublayers each of either different materials or the samematerials listed previously based on the function of said sublayerlaminated by either adhesives bonding, thermal bonding, ultrasonicbonding, or other methods known to those skilled in the art. The layersand sublayers presented are grouped solely for the purpose of ease ofunderstanding the embodiment being discussed. In the present exampleimplementation involving molecular processing, the materials should becompatible with the fluids and in cases in which molecular amplificationis to be performed, the materials should not introduce substancesinhibitory to such amplification (e.g. RT-PCR) or which interfere withdetection of target microbes, and should not be contaminated withnon-target analyte (e.g. non-target microbial cells) or nucleic acids.The materials should also not adsorb target molecules, reactants, andreagent components to an extent which will interfere with the process.Example plastic materials and plastic film materials include, but arenot limited to, polycarbonate, polypropylene, PET, and cyclic olefins.

The chamber openings 710 may be sealed with a membrane seal, a foil sealor a cap 697 following dispensing of the wash buffer and pretreatmentfluid into the diluent chamber and macrofluidic centrifugation chamberrespectively. The seals or caps may be bonded using methods andmaterials compatible with heat sealing, adhesive bonding, ultrasonicbonding. Alternatively, the chambers may be sealed prior to dispensingof these liquids and alternate ports may be provided for the purpose ofdispensing these liquids and these ports may be sealed following thedispense operation. The cap 697 may be molded, embossed, machined orrapid prototyped, and may be constructed from polycarbonate,polystyrene, PET, polyester or other material appropriate to its formand function.

FIG. 10B provides an exploded view of the integrated cartridge 700,illustrating the stack up of the layers broken down in FIG. 10C-K, whichillustrates the main elements of each component. Each of the chambers inthe first component 698 possesses holes which lead from the respectivechamber to the top plane 731 of first component 698, for connection tofluid paths, vents, valves, and injection ports which are all within anupper laminated layer 699. The upper laminated layer 699 also containsall the elements for electrical lysing, reverse transcription, PCR anddetection of PCR products as described previously.

Diluent chamber 704 is connected fluidically to the macrofluidiccentrifugation chamber 703 via a pair of holes 707 in layer 731 andfluidic conduit 709 in layer 732 and flow through this path iscontrolled by the valve 720, the membrane of which is on the top face of732. Supernatant chamber 705 is connected fluidically to macrofluidiccentrifugation chamber 703 via a pair of holes 708 in layer 731 andfluidic conduit 710 in 732 and fluid flow through this conduit iscontrolled by valve 721, the membrane 01 which is on the top face of732.

Diluent chamber 704 and supernatant chamber 705 also each possess a hole706 in layer 731, which respectively leads to a vent 740 on the upperlayer 737 via complementary holes in the intermediate layers.Macrofluidic centrifugation chamber 703 possesses hole 711 in layer 731which leads to an air injection port 741 on the upper layer 737. A pairof holes 712 in 731 and fluidic conduit 714 in 732 provide a fluidicconnection between the needle in the sample transfer receptacle 702 andmacrofluidic centrifugation chamber 703 in component 698, and flowwithin fluidic conduit 714 is controlled by valve 719, the membrane ofwhich is on the top face of 732.

The sample centrifugation and wash processes described above can beimplemented on integrated cartridge 700 by way of air displacement viaport 741, and the selective closing and opening of the various valves.Sample fluid may be directed from the sample tube 520 which is insertedin the sample transfer receptacle 702 via path 714 in layer 732.Subsequently, after centrifugation, supernatant from macrofluidiccentrifugation chamber 703 may be directed to the supernatant chamber705 via conduit 710 in layer 732, and diluent liquid may be directedfrom diluent chamber 704 to macrofluidic centrifugation chamber 703 viapath 709 in layer 732. Following the completion of the centrifugalseparation and wash process described above, valve 722, whose membraneis located on the top surface of layer 732 is opened and the final cellsuspension in macrofluidic centrifugation chamber 703 is displaced viahole 713 in layer 731 to collection chamber/conduit 715 in layer 732.

In the example embodiment shown, the displacement of the residual cellsuspension to and from macrofluidic centrifugation chamber occur by airdisplacement into and out of port 741, by way of an air displacementpump connected to port 741. Following the displacement of the residualcell suspension to collection chamber 715, subsequent displacements offluid occur by air displacement through port 743 in layer 737 by way ofan air displacement pump connected to port 743. In an alternativeembodiment fluid displacements continue to be activated by airdisplacement through port 741 and port 743 forms a vent.

Suspension chamber 715 is connected via a hole through intervening layer733 and 734 to fluid path 723 in layer 736 and subsequently throughvalve 724 which is located on the top surface of 736 to the electricallysing chamber 716, though the intervening holes on 735. The lysingchamber faces are constructed of surface enhanced oxidized electrodes asdescribed in US Patent Application No. US20120190040, titled “CELLCONCENTRATION, CAPTURE AND LYSIS DEVICES AND METHODS OF USE THEREOF” andfiled on Apr. 16, 2012, which is incorporated herein by reference in itsentirety, and in US Patent Application Publication No. US20140004501,and these electrodes are electrically connected through the interveninglayers to the terminals 747 in layer 737 exposed on the upper face ofthe cartridge. Electrical connection between the electrodes and therespective terminals (contacts) may be made by wire bonding, bonding ofa conductive element between the layers or sandwiching a conductiveelement between the layers.

Lysing chamber 716 in layer 734 is connected fluidically through valve725 in layer 736 to fluid path and lysate chamber 717. Dry formatreagents are optionally deposited on either the top or bottom face ofchamber 717. Lysate chamber 717 is connected to fluid conduit 726 inlayer 735, which leads to valve 727 in layer 736 and the network offluid paths and thermal chambers 728 in layer 736.

The bottom surface of chambers 728 may optionally be an air permeablemembrane layer 718 which allows flow of air or other gases, but notliquid, through the membrane to path 742 in layer 733 leading to port743. An example material is porous PTFE membranes or other materials.The top surface of 736 is a membrane which is optically transparent, forexample, to the excitation and emission spectra of the fluorophore dyesin PCR reagents, and sufficiently thin, to serve as the membranematerial for valve 724 and 725. Example materials for this purposeinclude, but are not limited to, polycarbonate, cyclic olefins, PET orother membranes films. In applications involving fluorescence detection,materials employed for the chamber side and bottom layers should beselected so as to minimize autofluorescence emission, which mayotherwise interfere with PCR signal detection. In the case of a PTFEmembrane which is typically white, layer 735 may be opaque, and features729 which make up the bottom of chambers 728 prevent the imaging of theunderlying white membrane in the center of the chamber, however allowthe passage of air around the outside perimeter of each chamber 728.

Dry reagents are optionally deposited on either to top or bottom surfaceof thermal chambers 728.

The top surface of the thermal chambers 728 in layer 736 contact thebottom surface 737 b of layer 737 which possesses an array of resistiveheaters 744 in a pattern matching the array of thermal chambers in layer736. Alternately the resistive heaters may be applied or printeddirectly on the top surface of the thermal chambers. The resistiveheaters are configured to heat the each chamber while allowing theoptical signal to pass through. For example, the individual chamberheaters may be in the form of a circular trace near the outsideperimeter of the chamber leaving a clear inner region for opticaltransmission as shown by 745

Likewise, a resistive heater 748 on 737 b may be in contact with orapplied to the top surface of lysate chamber 717 to optionally allowheating of that chamber. The resistive heaters are powered and themonitored via connection by the instrument (e.g. control and processingunit 140) to exposed terminals 746.

Layers 734, 735, 736, 737, and or 738 may have thermal properties whichallow dissipation of heat from the thermal chambers during the coolingphase of the PCR thermal cycles. Alternatively, a layer with propertiessuch as, for example, an aluminum foil layer, may be placed in closeproximity to the thermal chambers, for example layer 738 to dissipateheat for cooling purposes. This layer must have holes and cutouts toprevent interference with fluid and air paths described.

Thus the cell suspension in suspension chamber 715 is drawn into thelysing chamber 716 by opening all valves in the path to the port 743 andevacuating air from the fluid path through this port by way of an airdisplacement pump. For this action, valve 722 to macrofluidiccentrifugation chamber is also open and a path to atmosphere is providedfrom macrofluidic centrifugation chamber, via for example port 741.Alternatively the path to atmosphere from macrofluidic centrifugationchamber could be via one of the fluid paths (with valves open) to washvent or the waste vent 740.

In the present example implementation involving electrical lysis, thecell suspension is electrically lysed and treated by intermittentlyflowing a portion of the cell suspension into the chamber 716, closingthe valves 724 and 725, and applying the electrical train of bipolarpulses as described previously. According to the present exampleimplementation, electrical lysis is performed serially in order to avoidthe need to treat the full suspension at once, which reduces theelectrical current that is required. Valves 724 and 725 are then openedand a further volume of cell suspension is passed into the chamber 716thus displacing the previously lysed cell suspension to chamber 717. Avolume equal to the volume of the chamber 716 or optionally a portion ofthe full chamber volume may be passed into the chamber at eachsubsequent electrical lysing step to ensure that all of the cellsuspension is lysed during the sequence of lysing steps.

Lysing is complete after the full volume of cell suspension has beenpassed into the chamber 716 and the resulting lysate has been passedinto chamber 717. The lysate in the chamber 717 will dissolve thereagent which has optionally been placed on the bottom or upper surfaceof the chamber. The dissolving process can optionally be assisted byraising the temperature of the fluid to approximately 40° C. applied byheater 748. Optionally the lysate fluid may be passed back and forththrough the fluidic path comprising the lysate chamber 717, theelectrical lysis chamber 716 and the cell suspension chamber 715 and thefluidic paths, holes and valves in the fluidic path between these byalternate injection and evacuation of air through port 743. This actionmay promote dissolving of the dry reagents and also promotes lateral andlongitudinal mixing of the fluid via Taylor dispersion to increase thehomogeneity of the solution with respect to reagent components andtarget nucleic acids.

The lysate reagent solution is then passed through to the thermalchambers by drawing air through port 743. The negative pressure producedat the exit from the permeable membrane 718 in path 742 will promote theevacuation of air and air bubbles from the chambers. Valve 727 is thenclosed and a positive pressure is optionally applied a port 743. Dryreagents present on one or more of the surfaces of the chamber willdissolve to provide the primers and optionally other componentsnecessary for RT- PCR. The RT-PCR thermal protocol is then initiatedusing the heater 744 and the cooling methods described previously.

Optionally a valve is placed in the path to vent 743 and this valve isclosed with valve 727 prior to the initiation of RT-PCR heating andthermal cycling.

In an alternate embodiment, the path from the lysis chamber 716 leads toa lysate chamber which is formed in the cartridge component 698. Alyophilized bead placed in this chamber contains some or all of therequired reagents for RT and/or PCR. The volume of this chamber is ofsufficient dimensions to contain the bead and of sufficient volume tocontain the required volume of lysate. The lyophilized reagents are thendissolved and the solution is optionally mixed to promote dissolutionand homogeneity of the mixture. Additional volume may optionally beprovided in this lysate chamber so that the solution can be effectivelymixed via vortexing of the cartridge.

The actuation of valves and the application of air displacement pressuremay be performed over all process steps while integrated fluidicprocessing cartridge 120 is housed within centrifuge 110. However, inother embodiments, the system may include a separate housing forreceiving one or more integrated fluidic processing cartridges 120,where the separation housing is not configured as a rotor forcentrifugation, but does include a suitable cartridge interfacingmechanism for actuating the valves and controlling fluid flow withinintegrated fluidic processing cartridge. This separate housing may beemployed to control the actuation of fluids within the microfluidicdevice of integrated fluidic processing cartridge 120 during assaysteps, or other steps, that are performed post-centrifugation andpost-washing, thereby freeing centrifuge 110 to be able to process anadditional integrated fluidic processing cartridge during the subsequentprocessing of the first integrated fluidic processing cartridge.

In an embodiment where the valves 722, 721, 720 and 724 are to be closedprior to engagement within the receptacle , and cartridge interfacingassembly 130, captive plungers 739 are included and held within thecartridge by layer 738. Further detail for the valve operation aredescribed below. An example for this embodiment may be to preventmovement of fluids held within chambers 703, 704, or 705 from flowinginto component 699 with between each other via fluid paths 710, 709,714, or 715 during shipment of the cartridge 700.

Examples of Valves

The integrated fluidic processing cartridge depicted in FIG. 10 employs,as an example, diaphragm valves, as detailed in FIGS. 11A-C.

FIG. 11A depicts a diaphragm valve which is closed by application of anexternal plunger 605 on a membrane diaphragm 601, which thus appliespressure to the membrane 601 circumferential to the port (hole) 603 andseals the port 603 preventing flow in fluid path 600. FIG. 11B depictsthe valve in the open state, where no external force is applied downwardon plunger 605. FIG. 11C depicts the plan view of the diaphragm valveillustrating the sealing pressure zone 606. In the embodiment depicted,plunger 605 may be provided as a component of an actuator which acts onthe cartridge. Alternatively the plunger may be a component of theintegrated fluidic processing cartridge where plungers are held captivein the valve pocket by a membrane covering the valve pocket. In thiscase the captive plunger is acted upon by an external actuator whichdelivers the force necessary to close the valve and is provided eitherby the cartridge receptacle which is part of the motorized rotor of FIG.1 or by the cartridge interfacing assembly 130 of FIG. 1.

Microfluidic layer 602, having a lateral microfluidic channel formedtherein, is bonded to valve base layer 615 which together form thefluidic path 600, where microfluidic layer includes a valve seataperture 618 in fluid communication with the lateral microfluidicchannel, where the valve seat aperture is positioned over the port 603and extends through the microfluidic layer 602. Optionally, microfluidiclayer 602 can be comprised of multiple layers which may comprise the topand bottom walls of the fluid path, with the exception of the valve seataperture (valve cavity) 618, where the top surface is the membranediaphragm. The membrane diaphragm 601 is bonded to layer 602, andprovides to top surface of fluid path 600 within the valve seataperture. The valve membrane diaphragm 601 may optionally be furthersandwiched between layer 602 and outer layer 604. The membrane diaphragmmay also optionally be manufactured such that some or all of the layers602, 601 and 604 are a single part with no bonding required, for exampleby molding, micromachining, embossing or other methods known by thoseskilled in the art. Retraction of plunger 605, or sufficient relaxationof the force with which plunger 605 is applied to the valve, allowsfluid to flow between along fluid path 600 as shown in FIG. 11B. Thevalve geometry and membrane material may be selected by one skilled inthe art so that under the closure force the membrane does not rupture.Additionally, in the embodiment shown the valve plunger should besufficiently large to provide sufficient area to form a seal around port603. This may be at a minimum approximately 2 time the diameter of theport 603

In the embodiment of FIG. 11A, without the application of force to theplunger 605, the membrane will not seal the port 603 and fluid may flowalong fluid path 600 as shown in FIG. 11B. This embodiment is acceptablewhen the fluid path does not need to be closed prior to actuation orprior to engagement with the cartridge interfacing assembly. In manycircumstances it is necessary that some or all of the valves in theintegrated fluidic processing cartridge be closed in the absence of anactuator mechanism. For example it may be desired to have valves 720,721, 722, and 719 in FIG. 10D closed during handling, transportation andstorage of the cartridge to prevent fluids preloaded into macrofluidiccentrifugation chamber 703 and diluent chamber 704 from passing to otherchambers or fluidic paths prior to initiation of cartridge samplepreparation operations.

FIG. 11D illustrates another embodiment of the diaphragm valve which hasthe added feature of being closed without the application of an externalactuator. In this case, a captive internal plunger 613 is supplied whichis placed between an outer membrane 611 and the diaphragm membrane 601.The captive internal plunger may be bonded to the outer membrane 611and/or the membrane 601. Membrane 611 may be bonded to the membranelayer 601 or there may be additional layers between 601 and 611.Membrane 611 may optionally be sandwiched with a covering layer 604. Thecaptive plunger 613 is dimensioned such that it extends above the toplevel of layer 602 when the valve membrane 601 is in the closed positionand the membrane is applied such that within the valve seat aperture618, it is under a tensile stress sufficient to supply a reactivecompressive pressure to the captive plunger 613 which is sufficient toseal port 603 with the membrane diaphragm 601. This embodiment allowsthe cartridge to be transported, stored and handled without liquidtransfer between the chambers of the cartridge or between the chambersand the microfluidic backplane of the cartridge. This is particularlyuseful, for example, when a pre-treatment fluid is present in thecentrifugation chamber or when a wash diluent solution if present in thediluent chamber.

In one exemplary method the membrane is placed under tension, eitheruniaxial or biaxial and is placed over the captive plunger and bonded orsandwiched in place while the tension is maintained.

In order to open the valve to allow flow in flow path 600, a valveplunger actuator is provided external to the cartridge which can cut themembrane and thus release the tension in the membrane sufficiently torelieve the pressure between the captive plunger 613 and the valve baselayer 615. This device may be provided as a component of cartridgeinterfacing assembly 130 or the cartridge receptacle provided as part ofcentrifuge 110, thereby enabling robotic actuation when integratedfluidic processing cartridge 120 is loaded within centrifuge 110.

In one example embodiment, the valve plunger actuator 612 possesses acutter 616 on the perimeter of the plunger actuator 612 which uponengagement with the membrane in the gap between the plunger 613 and thevalve seat aperture 618 will cut the membrane upon the application of anadequate force as shown in FIG. 11F. Cutter 616 can extend around thefull circumference of the captive plunger 613 to fully cut the membrane,or the cutter 616 may extend partially around the circumference to cut aportion of the membrane 611 as depicted in FIG. 11E by the cutting line614. In the later embodiment the membrane tension may be partiallyreleased such that the plunger 613 remains captive but the pressurebetween the plunger 613 and the valve base 615 is relieved to an extentadequate to allow fluid flow between along fluid path 600.

In an alternative embodiment the tension on membrane 611 is uniaxial andmembrane 611 is cut only on that portion of the membrane under tensionand in a direction transverse to the uniaxial membrane stress. Thus thevalve plunger pressure is relieved but the plunger remains captive.Following relief of the valve plunger pressure in the manner describedin various embodiments above, valve closure may be reactivated byapplication of the valve plunger actuator surface 617 to the captivevalve plunger 613. Application of sufficient force to the valve plungerwill re-engage captive plunger 613 with the diaphragm and valve base andreseal the port 603 as shown in FIG. 11F. Retraction of the plungerrelieves the valve pressure and allows flow to occur in path 600 asshown in FIG. 11G.

FIGS. 11H and 11I illustrate two alternative implementations of thediaphragm valve. In FIG. 11. H, the lateral microfluidic channel doesnot extend over the full height of the microfluidic layer. In FIG. 11I,the second membrane is bonded to the top layer 607, instead of joined tothe first membrane.

In the embodiment shown in FIG. 10, an example valve membrane 601thickness is between 0.025-0.25 mm, preferably 0.075 to 0.125 mm and anexample fluid path 600 height is 0.025-0.5 mm, preferably 0.1-0.25 mm,and an example width is 0.1-4 mm. An example valve seat aperture 618diameter is 2-8 mm, preferably 3-6 mm, and an example port 603 diameteris 0.1-3 mm, preferably 1-2 mm. An example membrane 611 is an aluminumfoil of thickness between 0.025-0.2 mm.

Examples of Ports

Example integrated fluidic processing cartridge depicted in FIG. 10Bpossesses air displacement ports 741 and 743, which allow connection toan air displacement device to move fluids within integrated fluidicprocessing cartridge 700 as discussed previously. According to theexample embodiment shown in FIG. 10B, the ports are engaged anddisengaged with a removable air nozzle head 630 which is connected by atube or other air path to the air displacement device. The air nozzlemay be integrated into the cartridge interfacing assembly 130 such thatthe cartridge ports 741 and 743 may be engaged and disengaged when thecartridge interfacing assembly is engaged with the cartridge.

FIGS. 12 and 12B depicts an embodiment of such port 631 and an airnozzle head 630 which can be intermittently engaged and disengaged fromthe port. The nozzle head has an air path 633 connected to the airdisplacement device directly or via a rigid or flexible tube, and anozzle 632. Optionally, nozzle 632 has a beveled edge and the air nozzlehead has a face seal 634. The face seal 634 may be a rubber or othersoft material which can obtain a seal with the face 642 of the port 631.

Port 631 includes hole 636 formed in laminate layer 639, where hole 636is connected to air path 638 in layer 641. Optionally, a layer 640between the layers possesses an air permeable membrane 637. Also, porthole 636 may optionally be sealed by membrane 635 which is bonded tolayer 639 or sandwiched between the layer and an optional top layer 643.

Air nozzle head 630 is engaged with the port by punching seal 635 withthe air nozzle 632 (or with another suitable punching device) andbringing air nozzle head face seal 634 into contact with face 642 of theport and applying the necessary pressure to seal the interface betweenthe face of the seal 634 and the face 642 of the port. Air nozzle 632aligns with and enters hole 636 during this action. Optionally, membrane635 may be omitted such that the aforementioned punching action is notrequired. In such a case, air nozzle extension 632 from the body of theair nozzle head may optionally be omitted and the air path 633 broughtinto alignment with the hole 635 during engagement of the air nozzlehead with the port.

In another embodiment the face seal 634 may be omitted, and the seal maybe established between the face of the body of the air nozzle head andthe face 642 of the port if sufficient force is applied and thematerials used allows for a seal under these conditions. Membrane 635may be a metal foil, e.g. aluminum foil, or a plastic membrane, e.g.polycarbonate, polyimide, PET, polypropylene, cyclic olefin or othermaterial. Optional membrane 635 serves to provide a seal to the portprior to the first engagement with a connector nozzle, preventing theingress of liquids or contaminants into the port. Optional air permeablemembrane 637 serves to prevent passage of fluid from path 638 into theair nozzle head and optionally filters the air injected or evacuated bythe air displacement operation. Thus integrated fluidic processingcartridge 120 is protected from airborne contaminants or interferentswhich may otherwise enter the cartridge via the port, and airbornemicrobial cells are prevented from entering or exiting the cartridgethough the port. For this purpose, a membrane or other filter may beused for the element 637 which has a pore size of approximately 0.4microns or less.

Examples of Air Vents

Air vents are provided to assist fluid flow into and from otherwisesealed passages and chambers at various locations in the cartridge. Forexample, in the embodiment of FIG. 5, by providing a vent 518 toatmosphere, atmospheric pressure can be attained in the supernatantchamber 506 so that a positive pressure differential promoting fluidflow can be obtained along conduit 511 by applying positive pressure viaport 518 in centrifuge chamber 502 with the air displacement device. Thestructure of one example embodiment of an air vent is similar to theport of FIG. 12. When the optional pierceable membrane 635 is included,the vent is activated by piercing the membrane with a needle headequipped with a piercing needle to allow the passage of air.

Instrument/System

As described above, system 100, which may be provided as a benchtopinstrument, contains a centrifuge having a motorized rotor. Themotorized rotor is capable of speeds necessary to provide thecentrifugal sedimentation force necessary for a given application oruse, such as to sediment a wide range of target microbes in the fluidmedium.

The sedimentation occurs in macrofluidic centrifugation chamber 200 ofintegrated fluidic processing cartridge 120 and those skilled in the artcan determine the relationship between the rotor speed, rotor radius,cartridge geometry and centrifugation time necessary to sediment theparticles (e.g. microbial cells or other cells) with known sedimentationcoefficients. Sedimentation coefficients can be determined empiricallyby centrifuging target microbes in fluids of interest using commonlyavailable benchtop centrifuges and known methods for measuring recovery.

The centrifuge may be of the fixed angle type or the swinging buckettype and centrifuge parameters are adjusted accordingly.

An example embodiment of the centrifuge is shown in FIG. 13B in planview and in FIG. 13C in side view. The embodiment in FIGS. 13B and Cdepicts a swinging bucket centrifuge with rotor 801, two cartridgereceptacles 802 which swing on hinge pins 803, and drive motor and shaftassembly 804. The cartridge described previously is placed in thereceptacle and subjected to centrifugation at the appropriate steps inthe centrifugal separation and washing process described in FIG. 3.Under full speed centrifugal rotation the cartridge receptacle willswing to occupy the horizontal position 805 due to centrifugal forcesacting on the receptacle 802 and revert to the vertical orientation 807when rotation stops.

An example embodiment of a cartridge receptacle which accepts theintegrated fluidic processing cartridge, such as the cartridgeembodiment 700 depicted in FIG. 10, and provides the necessary interfaceelements for cartridge 700 is illustrated in FIG. 13A. In this exampleembodiment the cartridge is inserted from the top as shown and issecured in the receptacle that it is engaged with interface elements onthe receptacle which may include electrical contacts, fluidic ports,valve actuators, and optical module components. These interface elementsin turn engage with mating elements on the cartridge interfacingassembly 120 when the cartridge interfacing assembly engages with thecartridge receptacle so that the cartridge interfacing assembly cancontrollably actuate or activate the various elements as required.Alternatively, for some or all interface elements, access holes andareas may be provided to allow those interface elements present on thecartridge interfacing assembly to interface directly with the cartridge.In some embodiments interface elements on the cartridge receptacle areengaged with the cartridge only after the cartridge interfacing assemblyengages with the cartridge receptacle. Thus the cartridge interfacingassembly 130, controlled by the central control and processing unit 140,either directly, or indirectly through intermediate interface elementson the cartridge receptacle, acts on the cartridge to perform thevarious functional operations described in relation to the variousembodiments described and anticipated herein.

As shown schematically in FIGS. 14A and 14B, the cartridge interfacingassembly, here depicted schematically as 810, can be brought intoposition 814 and engaged with the face of cartridge receptacle 810. FIG.14A provides a plan view of the motorized rotor 801 and cartridgeinterfacing assembly 810 and depicts a position 811 to which thecartridge interfacing assembly 810 is retracted out of the path of therotor and swinging bucket during centrifugation. When the centrifugationceases, the centrifuge rotor is brought to rotational position 812 suchthat the cartridge interfacing assembly 810 can be brought into positionto engage with the cartridge and cartridge receptacle. This rotorpositioning action may be performed by the centrifuge drive motor eitherdirectly in conjunction with a position sensor, or by provision of abraking mechanism which stops the rotating rotor at a predeterminedposition. Alternatively a motorized positioning wheel may engage withthe rotor or rotor shaft after centrifugation has stopped and drive therotor to the required position. Position sensors may be provided toassist rotor positioning.

The cartridge interfacing assembly 810 must move into position andengage the cartridge receptacle and optionally the cartridge directlyfor various actions. The cartridge interfacing assembly 130 may be fixedto a translation stage and/or a rotational stage which give it thenecessary translational and/or rotational motions to move into positionlateral to the face of the cartridge and to engage the cartridgereceptacle. The cartridge interfacing assembly 130 may engage thereceptacle by latching it and holding it rigidly or semi-rigidly, or itmay come into contact with it and engage it with a fixed stop or bracketwhich will prevent the swinging action and lock the cartridge receptaclein place. The cartridge interfacing assembly contains the variousinterface elements necessary to perform the various actions necessaryfor the processes described herein with respect to the variousembodiments of the integrated fluidic processing cartridge. This mayinclude electrical connectors or contacts, actuators, fluidicconnectors, pumps, air displacement devices, optical devices and otherdevices which enable the required electrical, mechanical, fluidic andoptical operations to be performed. Some examples of these devices andcomponents and interface elements supplied on the cartridge receptacleare described below. These are intended to be representative of typicaldevices and elements required to perform the functions described hereinwith respect to various embodiments are provided below but is notexhaustive nor complete. Additional and alternative devices, componentsand elements may be determined by those skilled in the art.

A multi contact electrical connector, or multiple electrical connectors,may be employed to provide electrical power to the various cartridgeterminals and to transmit and/or receive electrical signals from someterminals to power the heating elements for reverse transcription andPCR, to detect temperatures by means described previously, and toprovide electrical power to the electrical lysing elements. Theelectrical connection may be made directly between a multi contactconnector on the cartridge interfacing assembly 130 and the cartridgeterminals via an opening in the receptacle when the cartridgeinterfacing assembly 130 is engaged with the cartridge. Alternatively anelectrical connection can be made between the cartridge terminals and amulti contact connector in the receptacle and upon engagement of thecartridge interfacing assembly 130 with the receptacle, a connector onthe cartridge interfacing assembly 130 makes electrical contact with therespective contacts or connector on the cartridge receptacle. Suchelectrical connections may be, for example, pogo pins, spring clipconnectors, contact probes, card connectors, PAD connectors, leaf springcontacts/connectors, compression connectors, cylindrical springcontacts, spring finger contacts, or other such electrical contactsknown to those skilled in the art.

The cartridge interfacing assembly 130 may include one or more airnozzle heads 630 which engage directly with the cartridge ports 631 onthe cartridge as described in relation to the embodiment in FIG. 12 oras may be required for other equivalent embodiments. The cartridgeinterfacing assembly 130 either contains an air displacement device oris connected by way of a flexible tube to an air displacement devicemounted in another fixed location in the instrument. The airdisplacement device may be a syringe pump, peristaltic pump.Alternatively the receptacle may contain an air nozzle head and thecartridge interfacing assembly 130 engages with this nozzle head toengage it with the cartridge and to effect the required airdisplacements. In some embodiments multiple air nozzle heads may bepresent to enable air displacement in additional cartridge ports. A ventneedle can similarly be present on the cartridge interfacing assembly130 and be directly engaged with the cartridge or alternately be mountedin the cartridge receptacle and engaged with the cartridge uponactuation by the cartridge interfacing assembly 130.

Valve Actuators

As depicted in FIG. 11A, the example valve actuation mechanism describedpreviously requires an actuator plunger to apply pressure directly tothe diaphragm 601 or to apply pressure to in intermediate captiveplunger (e.g. 613) in the cartridge assembly. This actuator plunger isin some embodiments mounted within the cartridge receptacle in a mannerwhich allows it to be engaged and actuated by the cartridge interfacingassembly 130 to effect required cartridge valve actions as well as suchactions as may be required to allow the cartridge to be inserted intothe cartridge receptacle. FIG. 15 provides example embodiments ofcartridge actuation mechanisms with schematic cut-out views of thecartridge and cartridge receptacle wall.

In FIGS. 15A-E, a schematic cross section view of a valve in cartridge820 is shown in relation example embodiments of actuator pins mounted incartridge receptacle 822. The valve is in open state. FIG. 15A-B showthe valve in open state and FIG. 15C-E show the valve in closed state.

In FIG. 15A a hole is provided in the wall 822 of the cartridgereceptacle in alignment with the valve which provides access for a pin819 mounted on an actuator on the cartridge interfacing assembly 130(not shown) to apply force to a captured plunger 825 on the cartridgethereby closing the valve as described previously with respect to FIG.11. Alternatively, in some embodiments, the captured plunger is omittedand pin 819 may contact the valve diaphragm 821 directly, therebyclosing the valve. Retraction of the actuator plunger relieves pressurefrom the diaphragm and fluid may be flowed with the application of anappropriate pressure differential along the flow path by the airdisplacement device connected to a cartridge port as described above.

FIG. 15B depicts an example of a pin 818 captive in the wall 822 of thereceptacle and optionally equipped with a spring to retract the pin.Thus the cartridge may be easily inserted into the receptacle withoutinterference and the valve will be in an open position when the actuatorpin 819 is not acting on it. The valve is closed when actuator pin 819,or some other similar element, applies a compressive force axial to thepin 818 such that it is brought into contact and applies pressure to thecaptive plunger 825 or optionally the valve diaphragm 821 directly.

FIG. 15C shows a pin 835 captive in the wall of the cartridge receptacleand optionally equipped with a pre-compressed spring 836 which acts onthe pin 835 to close the valve by applying compressive force to captiveplunger 825, or alternatively directly to the valve diaphragm 821. Thespring pre-compression should be sufficient to hold the valve closedunder all conditions under which that is required, including,optionally, during centrifugation of the integrated fluidic processingcartridge and receptacle. Thus, under no external actuation the valvewill be latched closed. External actuation is provided by an actuator onthe cartridge interfacing assembly 130 to retract the pin 835 andrelease the compressive force on the diaphragm valve to open the valveand allow fluid to flow. Such actuation may also optionally retract thepin 835 to allow clearance for inserting the cartridge into thereceptacle. Such actuation must retract the pin 835 against the springforce provided by spring 836 and may be accomplished in a number ofways. The cartridge interfacing assembly 130 may include a grippingmechanism to grasp the head of the pin 835 and pull the pin axially andin a direction away from the cartridge. Alternatively, a lever mechanism837 may bear against the surface of the cartridge receptacle and theunderside of the head of the pin which when actuated may lift the headof the pin and thereby retract the pin for the above purposes. Thisembodiment allows the valve to be latched closed such that closure ismaintained when the cartridge interfacing assembly is disengaged fromthe cartridge receptacle. Thus, during centrifugation, fluid will beprevented from flowing through such actuated valves. Note that thespring force must be sufficient to prevent leakage through the valveunder the substantial fluidic pressure which may occur in the valveduring high speed centrifugation

In FIG. 15D, the receptacle wall contains a threaded hole or threadedinsert which contains a screw 823 whose end face may be brought intocontact with the valve diaphragm 821 directly or into contact with acaptive plunger 825 on the cartridge as shown. Upon the application of asufficient amount of pressure the diaphragm valve will be closed. Thescrew 823 may be retracted to open the valve and to provide clearancefor insertion of the cartridge. Optionally a contact pin may be providedas a separate component and mounted in the receptacle wall intermediateto the valve and screw and optionally keyed in a manner so as to preventrotation of the pin as it is engaged and actuated by the screw 823. Thisembodiment also allows the valve to be latched closed such that closureis maintained when the cartridge interfacing assembly 130 is disengagedfrom the cartridge receptacle. Thus during centrifugation fluid will beprevented from flowing through such actuated valves. Note that the valveactuation force must be sufficient to prevent leakage through the valveunder the substantial fluidic pressure which may occur in the valveduring high speed centrifugation.

FIG. 15E provides another example embodiment where a lever 826 withhinge pin 828 is mounted in or on the receptacle wall. The lever 826 isoptionally equipped with a pre-compressed spring 827 which applies asufficient force to the lever such that it maintains contact with thecaptive plunger 821 and closes the valve.

Note that in some alternate embodiments the plunger may optionally beequipped with a retraction spring which acts to release of pressure fromthe valve force.

The valve may be acted on by an actuator 832 on the cartridgeinterfacing assembly to cause counterclockwise rotation of the lever826. In doing so the actuator overcomes the spring force and releasesthe force applied to the plunger 825. The pressure is thus released fromthe valve to allow fluid flow. When the lever is released by theactuator the lever assumes the valve closing position assisted by spring827. This force must sufficient to maintain leak free closure of thevalve at rest and optionally during the operation of the centrifuge. Inanother embodiment the center of mass 830 of the lever 826 ispreferentially positioned such that under centrifugal rotation for whichcentrifugal forces are in the direction 831, the centrifugal forceexperienced by the lever creates a compressive reactive force on theplunger 825 acting to further increase the force applied to the valve.The additional valve closure force produced in this manner may providethe assistance required to seal the valve even under the high fluidicpressures which may be experienced at high centrifugal speeds. For thisembodiment the spring 827 closure force need only be sufficient for leakfree closure of the valve up to centrifugal speeds where the centrifugalforce exceeds the spring force.

In all of the embodiments depicted in FIG. 15, the surface which bearson the captive plunger on the cartridge may be optionally equipped witha cutter 616 as depicted in FIG. 11. Actuators which perform theactuations described may take one or more of many different formsincluding solenoids, hydraulically actuated pistons, servos, DC motors,and stepper motors. Linear actuators may incorporate the actuator pins819 and 832 directly, or they may act via an intermediary mechanismwhich includes actuator pins 819 and 832, or lever 837 or they may actthrough an intermediary mechanism which converts linear motion torotational motion as for actuator screw 823. Rotational actuators suchas servos, DC motors and stepper motors for example, may act directly onthe actuator screw 823, incorporating an engagement mechanism whichallows the actuator screw 823 to be engaged, rotated as required, anddisengaged. Such rotational actuators may also be used for linearactuation of actuator pins 819 and levers 826 and 837 via anintermediary mechanism such as cams, levers or other mechanisms whichconvert rotational motion to linear motion.

Mixing

Example embodiments for mixing fluids in the chambers in the integratedfluidic processing cartridge are provided. Cyclic cartridge inversion isan effective mixing method whereby the cartridge is rotated from anupright position to a fully inverted position or a partially invertedposition, and then back to the initial upright position in one inversionmixing cycle. The swinging bucket receptacle allows this action byextending the swing path to an inverted position such as depicted byposition 815 in FIG. 15A and B. For example, the cartridge interfacingassembly 130 can be used to actuate this motion by taking a position 813to the side of the cartridge receptacle and engaging the receptaclewhile remaining free of the swing path. In one embodiment the cyclicswing action can be actuated using an arm which engages with thecartridge receptacle and moves the receptacle through its range ofmotion by the way of a DC motor, stepper motor, solenoid or servo.Alternately a gear interface may be provided between a rotary drivedevice on the cartridge interfacing assembly 130 and the receptacle.

An embodiment which allows for fluid mixing (e.g. fluid agitation) inthe cartridge by vortexing of the cartridge is now described. Forexample, the vortexing action may be an orbital displacement of the baseof the cartridge (816 in FIG. 14B) in the plane of the base. Forexample, the orbit may be 5 mm and the orbiting speed may be 1000 rpm.This action can be performed by engaging the bottom of the cartridgereceptacle with a motor driven rotary element on the cartridgeinterfacing assembly. The rotary element may be a cam which contacts afeature on the bottom of the cartridge receptacle, or a disc with anoffset pin engaged with the bottom of the cartridge. For example, FIG.16 shows a bottom view of the cartridge receptacle 802 with rotaryelement 850. The eccentric engagement point 852 between rotary element850 and receptacle 816 is offset from the center of rotation 851 of therotary element 850 such that as the rotary element 850 rotates, thecartridge receptacle bottom 816 follows the circular orbit 853. Thus fora 5 mm diameter radius the engagement point is offset 2.5 mm from thecenter of rotation of the rotary element. The engagement between therotary element and the cartridge receptacle may occur via a bushing, abearing or otherwise rotationally free junction. Alternately aneccentrically driven rotary element can be brought into contact with thebottom of the cartridge receptacle and by friction or another means ofengagement so that the bottom of the cartridge may be rotated throughthe desired orbit. While the bottom of the cartridge is orbited in thisway, the top of the cartridge receptacle must possess sufficient freedomto allow this motion to occur. The swinging motion (shown as 806 in FIG.14B) of the receptacle 802 about the hinge 803 provides freedom for thecomponent of orbital motion in the direction of the rotor radius. Thecomplementary component of the orbital motion is in the rotorcircumferential direction and may be accommodated by providing a hinge(803 in FIG. 13A) which allows such motion. For example, the swingingreceptacle hinges 803 may be engaged in vertical slots on the respectiveside walls of the receptacle allowing the require rocking motion of thecartridge which conforms to the circumferential component of the orbitaldisplacement at the cartridge base. Alternatively the vortexing motionimparted to the bottom of the cartridge receptacle may be linear in theradial direction causing an alternating swinging action 806 whichconforms to the swinging motion provided by the cartridge receptaclehinge. Such action may be produced by the rotating element 850 in whichthe lateral component of the motion is released by a sliding mechanismin the engagement mechanism with the cartridge receptacle or within therotary element itself. Alternatively this may be produced by some otherrotational to linear motion mechanism such as a cam or lever, or byactuation with a linear actuator.

Example of Cartridge Receptacle and Cartridge Interfacing Assembly

An example embodiment of a cartridge receptacle 900 is provided in FIG.18A FIG. 18B provides an example embodiment of a cartridge interfacingassembly 950 which, as depicted in FIG. 18C, is able to be translatedinto position by a robotically controlled translation stage (not shown)and engage with the cartridge receptacle when the motorized rotor 951has come to rest in the rotational position which conforms with thealignment requirements of the engagement of the cartridge interfacingassembly, and the cartridge receptacle assumes a vertical position. Thecartridge interfacing assembly, when translated into position to engagethe cartridge receptacle, may press against the cartridge receptacle toengage the cartridge receptacle with stops on the opposing side (notshown) so that the cartridge receptacle is restrained from swinging awayfrom the cartridge interfacing assembly and is held firmly as thecartridge interfacing assembly engages with the cartridge receptacle.Alternatively the cartridge interfacing assembly may include a mechanismwhich is able to secure the cartridge receptacle and hold it in positionfor engagement.

The example cartridge receptacle 900 is a swinging receptacle with hingereceptacles 901 which engage with the rotor pins. The sample cartridge904 is shown inserted into the cartridge receptacle. The receptaclefurther includes actuator pins 902 which are held captive in thereceptacle wall shown (in cross-section) as 935 in FIG. 17A and whichare equipped with a pre-compressed spring 930 and a head 931 whichprotrudes from the surface of the cartridge receptacle. This actuatorpin embodiment is the latched closed embodiment of FIG. 15C which holdsall valves closed by means of the spring force when the cartridgeinterfacing assembly is not engaged and when the actuator lever is notactuated. The protruding head 931 allows engagement with a lever 932hinged at 933 and mounted in the front face 937 of the cartridgeinterfacing assembly 950.

FIG. 17A shows the cartridge interfacing assembly 950 in the engagedposition with the cartridge receptacle 900 and where the lever hasengaged with the actuator pin head 931 protruding from the cartridgereceptacle . In the lever position of FIG. 17A the actuator pin remainsin unactuated position and the actuator pin 902 is acted upon solely bythe pre-compressed spring 930 and against the valve 936 and the valvediaphragm is in a closed position. The lever has a notched feature whichallows it to engage with the protruding head 931 as the cartridgeinterfacing assembly is brought into close proximity to the cartridgereceptacle. For example, the lever may be rotated clockwise into theposition of FIG. 17A as the cartridge interfacing assembly approachesthe cartridge receptacle such that the notched feature 942 engages withthe narrowed region 940 of protruding head 931 as shown in FIG. 17C.FIG. 17B shows the lever in an actuated position which opens valve 936by clockwise rotation of lever 932 about hinge 933 so that the levernotch 942 contacts the widened top portion 941 of the actuator pin head931 so that actuator pin 902 is raised thus releasing the pressure onthe valve 936.

The cartridge receptacle further includes access holes 904 for airnozzle heads 959 to engage directly to cartridge ports 741 and 743 (ofFIG. 10). The air nozzle pins are optionally spring mounted on cartridgeinterfacing assembly to allow the sealing face of air nozzles 959 tocontact the cartridge port and apply a compressive force between saidface and port. The spring stiffness and an optional spring perforce maybe prescribed which will ensure that sufficient force is applied so thata seal can be repeatedly be made which will withstand the pressureapplied to the port during fluid transfer. Access holes 905 are providedfor the electrical lysing contact pins 952 to make electrical contactwith the cartridge electrical lysing terminals 747. Such electricalcontact pins may be spring loaded pogo pins to ensure reliable contact.The array of electrical contacts 906 are provided on the cartridgereceptacle surface for connection to a mating array of terminals or pins953 on the cartridge interfacing assembly. The contacts 906 areconnected electrically to terminals within the cartridge receptaclewhich are spring loaded or otherwise configured so that, upon insertionof the cartridge into the cartridge receptacle, they make electricalcontact with the contacts 746 on the sample cartridge. This electricalconnection provides the means by which the cartridge heaters are poweredand monitored. The cartridge also has access holes 910 which allowoptional vent piercing pins on cartridge interfacing assembly 950 toreach and pierce an optional vent membrane seal on the cartridge ventsupon engagement of the cartridge interfacing assembly. Cartridgereceptacle 900 also has an access hole 908 to allow optical access tothe optical windows of the PCR chamber array 909 by the imager 954 orother optical module mounted on cartridge interfacing assembly 950.

Cartridge interfacing assembly 950 is shown with a camshaft 955 equippedwith multiple individual cams 956, each aligned with one of the valvelevers 932. The camshaft shown is driven by belt and pulley 957 and astepper motor 958 which can controllably position the camshaft atrotational positions for which the cam lobes come into contact with therespective lever arm 932 and to actuate the lever so as to open thevalve as described above. Each cam may have one or more lobes to enableactivation of its respective lever in one or more rotational positionsrespectively. In this way valves can be either actuated individually orin groups. For example, with reference to the embodiment of FIG. 5,during extraction of supernatant from the centrifuge chamber, valves509, 512, and 517 must remain closed and supernatant valve 513 opened aspositive gauge pressure is applied to centrifuge chamber port 518. Theremaining valves in the microfluidic backplane may optionally remainclosed during this operation. Thus a single cam on camshaft 955associated with the supernatant valve on the cartridge may actuate therespective lever on the cartridge interfacing assembly to open saidvalve as air pressure is delivered to the centrifuge chamber port whileall other valves remain unactuated and closed. In another operation, forexample, with reference to FIG. 8, valves 517, 565, 566, 567 and 572must be open to draw fluid from lysate chamber 562 to the PCR array 563by air evacuated from port 571. Thus, cam lobes corresponding to all ofthese valves, if present on the cartridge embodiment, must contact therespective valve levers to open all of these valves at the samerotational position so as to effect simultaneous opening of the valves.Individual cams may therefore have one or more lobes to allow actuationof the respective valve alone or together with other valves as requiredby the cartridge process. In some circumstances it may be necessary tohave more than one camshaft to accommodate complex valve processes.

The specific embodiments described above have been shown by way ofexample, and it should be understood that these embodiments may besusceptible to various modifications and alternative forms. It should befurther understood that the claims are not intended to be limited to theparticular forms disclosed, but rather to cover all modifications,equivalents, and alternatives falling within the spirit and scope ofthis disclosure.

1. A system for performing centrifugal separation, said systemcomprising: a fluidic cartridge comprising a valve; and a centrifugationdevice comprising: a rotor; and a receptacle pivotally connected to saidrotor, said receptacle being configured to receive said fluidiccartridge; said receptacle comprising a mechanical valve latchingmechanism capable of mechanically latching said valve in a closed stateduring centrifugation of said fluidic cartridge.
 2. The system accordingto claim 1 wherein said valve and said mechanical valve latchingmechanism are configured such that leakage of fluid through said valveis prevented when said valve is mechanically latched in the closed stateduring centrifugation despite fluidic pressures generated duringcentrifugation.
 3. The system according to claim 1 wherein saidmechanical valve latching mechanism is configured such that duringcentrifugation, a centrifugal force applied to said mechanical valvelatching mechanism results in a compressive force applied to said valve.4. The system according to claim 1 wherein said mechanical valvelatching mechanism comprises a retractable pin that is held captive in awall of said receptacle, said pin being biased against said valve whensaid fluidic cartridge resides within said receptacle.
 5. The systemaccording to claim 1 wherein said mechanical valve latching mechanismcomprises a spring-loaded assembly configured to hold said valve closedby a spring force when said fluidic cartridge resides within saidreceptacle.
 6. The system according to claim 1 wherein said mechanicalvalve latching mechanism comprises a ratchet device capable of lockingsaid valve in the closed state when said fluidic cartridge resideswithin said receptacle, wherein said ratchet device being releasable toopen said valve.
 7. The system according to claim 1 wherein saidmechanical valve latching mechanism comprises a screw that is receivedwithin a threaded aperture in said receptacle, wherein a position of anend face of said screw, relative to said receptacle, is adjustable tobring said valve into the closed state when said fluidic cartridgeresides within said receptacle.
 8. The system according to claim 7wherein said mechanical valve latching mechanism further comprises acontact pin mounted on said receptacle at a location intermediate tosaid valve and said screw, said contact pin being configured to beengaged and actuated by said screw during rotation of said screw forcontacting and closing said valve.
 9. The system according to claim 8wherein said contact pin is keyed in a manner so as to prevent rotationthereof during engagement and actuation by said screw.
 10. The systemaccording to claim 1 wherein said valve and said mechanical valvelatching mechanism are configured such that leakage of fluid throughsaid valve is prevented when said valve is mechanically latched in theclosed state during centrifugation with an applied centrifugal forcebetween 1000 g and 15,000 g despite fluidic pressures generated duringcentrifugation.
 11. The system according to claim 1 wherein said valveand said mechanical valve latching mechanism are configured such thatleakage of fluid through said valve is prevented when said valve ismechanically latched in the closed state during centrifugation with anapplied centrifugal force between 3000 g and 10,000 g despite fluidicpressures generated during centrifugation.
 12. The system according toclaim 1 wherein said valve and said mechanical valve latching mechanismare configured such that leakage of fluid through said valve isprevented when said valve is mechanically latched in the closed stateduring centrifugation with an applied centrifugal force between 2000 gand 12,000 g despite fluidic pressures generated during centrifugation.13. The system according to claim 1 wherein said valve and saidmechanical valve latching mechanism are configured such that leakage offluid through said valve is prevented when said valve is mechanicallylatched in the closed state during centrifugation with an appliedcentrifugal force between 4000 g and 8000 g despite fluidic pressuresgenerated during centrifugation.
 14. A method of performing centrifugalprocessing of a fluidic cartridge, the fluidic cartridge comprising avalve, the method comprising: providing a system according to claim 1,wherein the fluidic cartridge resides within said receptacle; andcontrolling the centrifugation device to centrifuge the fluidiccartridge while the valve latching mechanism maintains the valve in theclosed state.
 15. The method according to claim 14 wherein the valve andthe mechanical valve latching mechanism are configured such that leakageof the fluid through the valve is prevented when the valve ismechanically latched in the closed state during centrifugation despitefluidic pressures generated during centrifugation.
 16. The methodaccording to claim 15 wherein the mechanical valve latching mechanism isconfigured such that during centrifugation, a centrifugal force appliedto the mechanical valve latching mechanism results in a compressiveforce applied to the valve.
 17. A system for performing centrifugalseparation and fluidic processing, said system comprising: a fluidicprocessing cartridge comprising a valve; a centrifugation devicecomprising: a rotor; and a receptacle pivotally connected to said rotor,said receptacle being configured to receive said fluidic processingcartridge, said receptacle comprising a mechanical valve latchingmechanism; and a cartridge interfacing assembly capable of engaging withsaid receptacle and actuating said valve latching mechanism to open saidvalve when said rotor is at rest and said fluidic processing cartridgeresides within said receptacle; said valve latching mechanism beingconfigured to mechanically latch said valve in a closed state when saidfluidic processing cartridge resides within said receptacle and saidcartridge interfacing assembly is disengaged from said receptacle; andsaid mechanical valve latching mechanism comprising a pin that is heldcaptive in a wall of said receptacle, said pin being biased against saidvalve when said fluidic processing cartridge resides within saidreceptacle and said cartridge interfacing assembly is disengaged fromsaid receptacle, wherein said cartridge interfacing assembly is capableof retracting said pin to open said valve.
 18. The system according toclaim 17 wherein said cartridge interfacing assembly comprises agripping mechanism configured to grip said pin for retraction of saidpin.
 19. The system according to claim 17 wherein said cartridgeinterfacing assembly comprises a lever mechanism, and wherein saidcartridge interfacing assembly is capable of retracting said pin to opensaid valve.
 20. The system according to claim 19 wherein said levermechanism is configured to bear against a surface of said receptacle andan underside of said pin.
 21. The system according to claim 19 whereinsaid cartridge interfacing assembly comprises a camshaft, said camshaftcomprising a cam, wherein said camshaft is rotatable such that said camis contacted with said lever mechanism for opening said valve.
 22. Thesystem according to claim 19 wherein said lever mechanism comprises alever positioned such that during centrifugation, a centrifugal forceapplied to said lever results in a compressive force applied to saidvalve.